Humanized monoclonal antibodies and methods of use

ABSTRACT

The present invention comprises a humanized monoclonal antibody that binds to the chemokine receptor CCR4. This antibody is derived from Mab 1567 and recognizes the same epitope. Binding of the invented antibody to CCR4 inhibits ligand-mediated activities and is used to treat symptoms of cancer. Moreover, the antibody is used in combination with vaccines to suppress the activity of regulatory T cells.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/597,044 filed Jan. 14, 2015, now issued as U.S. Pat. No. 9,416,169,which is divisional of U.S. patent application Ser. No. 12/810,888,filed Mar. 11, 2011, now issued as U.S. Pat. No. 8,962,806, which is anational stage application, filed under 35 U.S.C § 371, of InternationalPatent Application No. PCT/US2008/088435, filed Dec. 29, 2008, whichclaims priority benefit of U.S. Provisional Patent Application No.61/017,494, filed Dec. 28, 2007, the contents of which are hereinincorporated by reference in-their entireties

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under grant numberCA093683 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to humanized anti-CCR4 antibodies aswell as to methods for use thereof.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “DFCI_048C01US_Sequence.txt”, whichwas created on Aug. 12, 2016 and is 12 kilobytes in size, are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Cutaneous T cell lymphomas (CTCLs) are the most common extranodalnon-Hodgkin's T cell lymphomas in adults. A recent WHO-EORTC consensusclassification (Willemze R. et al. Blood 2005, 105:3768-3785) indicatesthat there are thirteen clinically and histologically distinct types ofCTCL; however, 90% of CTCLs fall into three classes; mycosis fungoides,primary cutaneous anaplastic large cell lymphoma, and Sezary syndrome.The most common type of CTCL, mycosis fungoides, is characterized byerythematous patches and plaques that most commonly contain CD4⁺ T cellsthat show an affinity for the epidermis, or epidermotropism (Willemze R.et al. Blood 2005, 105:3768-3785). Staging is based upon a TNMclassification; patients with Stage 1A disease have normal lifeexpectancies, while patients with Stage 1B or greater have a diminishedlife expectancy (Kim, Y. H. et al. Arch Dermatol 2003, 139:857-866).Patients with Stage II-IV disease have a median survival of less thanfive years, with large cell transformation often leading to accelerateddeterioration (Kim, Y. H. et al. Arch Dermatol 2003, 139:857-866).Sezary syndrome is a leukemic variant of CTCL wherein clonal CD4⁺ Tcells accumulate in blood and lymph nodes as well as skin; five yearsurvival is less than 25%. Primary cutaneous ALCL has a much lessaggressive course, with a five year survival of 95%; however, cutaneousALCL with concurrent nodal involvement is more aggressive (Willemze R.et al. Blood 2005, 105:3768-3785; Kadin M E, Carpenter C. Semin Hematol2003, 40:244-256).

There is significant immune dysfunction in these patients, with globaldysregulation of the T cell repertoire of unknown etiology (Yamanaka K.et al. Clin Cancer Res 2005, 11:5748-5755; Yawalkar N. et al. Blood2003, 102:4059-4066). The terminal event in most patients is bacterialsepsis. Current therapies for advanced MF and Sezary syndrome arepalliative and durable long-term remissions are rare (Querfeld C. et al.Curr Opin Hematol 2005, 12:273-278). There is an urgent need for moreeffective therapies.

SUMMARY OF THE INVENTION

The invention is based upon the discovery of monoclonal antibodies whichbind the CC-chemokine receptor 4 (CCR4). The monoclonal antibody isfully human. In various aspects, the monoclonal antibody is a bivalentantibody, a monovalent antibody, a single chain antibody or fragmentthereof. Exemplary monoclonal antibodies include a monoclonal antibodythat binds to the same epitope as murine 1567.

The monoclonal antibodies of the invention can have a binding affinitythat is 1 nM⁻¹ or less. The monoclonal antibodies of the inventionfunction to inhibit viral and cell membrane fusion.

The monoclonal antibody has a heavy chain variable amino acid sequencecontaining SEQ ID NOS: 2, and/or a light chain variable amino acidsequence containing SEQ ID NOS: 4. The monoclonal antibody has a heavychain variable nucleic acid sequence containing SEQ ID NOS: 1, and/or alight chain variable nucleic acid sequence containing SEQ ID NOS: 3.

Also provided by the invention is an monoclonal anti-CCR4 proteinantibody or fragment thereof, where the antibody has a V_(H) CDR1 regionhaving the amino acid sequence GYTFASYY; a V_(H) CDR2 region having theamino acid sequence WINPGNVNTKYNEKFKG; a V_(H) CDR3 region having theamino acid sequence STYYRPLDY; V_(L) CDR1 region having amino acidsequence KSSQSILYSSNQKNYLA; a V_(L) CDR2 region having the amino acidsequence WASTRES and/or a V_(L) CDR3 region having the amino acidsequence HQYLSSYT.

In another aspect, the invention provides a method of selectivelykilling a tumor cell, e.g. a T cell tumor, by contacting a cell with ananti-CCR4 antibody according to the invention. The selective killingoccurs by antibody-dependent cellular toxicity (ADCC),complement-dependent cytotoxicity (CDC), or antibody dependent cellular.

Also included in the invention is a method of augmenting an immuneresponse to an antigen, e.g. a viral antigen such as HIV, a bacterialantigen or a tumor associated antigen by contacting a cell with ananti-CCR4 antibody according to the invention. The antibody isadministered prior to or after exposure to an antigen. The cell is aT-cell such as an effector T-cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are schematics showing Fluorescence Activated Cell Sorter (FACS)analysis of the expression of Cutaneous Lymphocyte Homing Receptors byCTCL cells identified by expanded TCR-Vβ-specific clone showing thatCTCL clones from leukemic CTCL patients are enriched in the expressionof homing receptors normally found only on cutaneous memory CD4 cells.

FIG. 2 are schematics showing FACS analysis of CTCL cells from leukemicCTCL patient and CTCL cells from skin lesion. The correlation betweenCLA loss (from 72% in skin to 27% in blood) and appearance of CTCL cellsin the blood (demonstrated by the presence of CCR4⁺ cells in the blood)suggests the CLA loss itself may contribute to the evolution fromlocalized CTCL lesions to full-blown leukemic CTCL.

FIG. 3 are bar charts showing CTCL Cell Migratory Response RecapitulatesThat of Normal Cutaneous T Cells but Not Normal Nave T Cells. In vitrochemotaxis assays demonstrate that CTCL cells display chemotacticmigration in response to the CCR4 ligand CCL22 and the CCR7 ligand CCL21(see panel A). Cutaneous memory cells display a similar pattern (seepanel C), whereas nave CD4 cells respond only to CCR7 ligand (see panelB). Data is mean±SD, 3 separate experiments, each experiment performedin duplicate.

FIG. 4 are schematics showing FACS analysis of T cells isolated from theskin lesions (see panel A) and blood (see panel B) of a patient withstage IV CTCL. A unique high scatter population of T cells was presentin both skin and blood; these cells represent a clonal population ofmalignant T cells. Malignant T cells from skin expressed both CLA andCCR4 (not shown) and malignant cells from blood expressed CCR4, but CLAexpression varied.

FIG. 5 is a bar chart showing Anti-Tumor Effect of Anti-CCR4 Mab 1567 ina CCR4⁺ Human ALCL Tumor Xenograft Model. Mab treatment was initiated onday zero (d0) by i.p injection of 3 mg/kg Mab and continued 2 times perweek for 5 weeks. Tumor size and growth were estimated by weeklymeasurement of tumor length and width by caliper photography. Resultsshow that from seven days post-injection of Mac-1 cells and onward,control tumors continued to grow while mice treated with Mab1567experienced reduced and arrested tumor growth. In mice receiving theMab1567 antibody, a tumor formed, but the size of that tumor remainedconstant over the next two weeks through post-injection day 21.

FIG. 6 are schematics showing FACS analysis of binding of 1567scFv/scFvFc. The scFv gene of anti-CCR4 Mab 1567 was cloned into a phagedisplay comprising either vector or Human IgG1 Fc-tagged expressingvector to express Mab 1567 scFv-phage and scFvFc. The specific bindingof these two types of antibody to CCR4 was examined by FACS using CCR4⁺cell lines 293T-CCR4 and Mac-1 cells. Filled blue areas representNegative scFv-phage or negative scFvFc control. Open, solid greenoutlined areas, represent Mab 1567 scFv-phage or scFvFc.

FIG. 7 are photographs of a Western Blot showing the expression ofCCR4-Nt-Fc fusion protein and epitope mapping of Mab 1567.

Left Panel, 293T cells expressing F105-L3-Nt-hCCR4-Fc fusion proteinwere labeled with [³⁵S]-cysteine and [³⁵S]-methionine (panel A) or[³⁵S]-sulfate (panel B). Secreted proteins contained within the culturesupernatant were immunoprecipitated with protein A sepharose beads andapplied to a SDS-PAGE reducing gel for analysis. Lane 1 and 2, WT orDDDD mutant version of CCR5 Nt Fc fusion protein (negative controls);Lane 3, human CCR4 Nt-Fc. Middle Panel (panel C), silver staining ofsecreted CCR4-Nt-Fc on non-reducing (lane 1) and reducing (lane 2) gelshowing the presence of dimeric and monomeric protein, respectively,with protein sizes demarcated by the marker (M) lane. Right Panel (panelD), ELISA with plate bound Mab 1567 and analysis of Mab 1567 binding tothree different Fc fusion proteins (PS11 scFvFc, CCR5-Nt-Fc, andCCR4-Nt-Fc) or BSA at three different concentrations (0.25 μg, 0.5 μg,or 1.0 μg per well). Binding detected by horseradish peroxidase(HRP)-anti-human Fc IgG.

FIG. 8 is a bar chart showing the inhibition of CCL22-Induced Mac-1 CellChemotaxis by Mab1567 and scFv1567-hFc. 1004 of Mac-1 cells (2×10⁵cells) were incubated with indicated concentration. Chemotaxis of humanATCL tumor cells, Mac-1, induced by the CCR4 ligand CCL22, is inhibitedby both Mab1567 and the scFv1567-hFc at very low antibodyconcentrations. Mab1567 (long/mL & 20 ng/mL), 1567scFvhFc, or an IgG2bisotype control were placed in the upper chamber of a 12-well transwellplate (of 5 μm pore size) while CCL22 (100 nM) was placed in 600 μL ofmedia in the lower chamber at 37° C. for 4 hours. Cells migration intothe lower chamber were determined by a FACS cell count using Flow-CheckFluorospheres as a reference (trials performed in duplicate).

FIG. 9 is bar chart showing ADCC activity of 1567scFvFc. The CCR4 scFvFc(R4) and HA-1 scFvFc (38B) antibodies were used at concentrations of 1ug/ml and 5 ug/ml, separately. Ratios of effector cells (PBMC) to targetcells (Mac-1) (50:1, 25:1, 12.5:1) were used. Trials were performed intriplicate wells in a 96-well plate.

FIG. 10 is a bar chart showing ADCC activity of 1567IgG and 1567scFvFc.The target Mac-1 cells were pre-labeled with APC dye TFL4 (OncoImmunin,Inc.) for 1 hour at 37° C. The effector cells (PBMC) to target cellswere mixed at ratios of 50:1 and 25:1, and centrifuged. The cell pelletswere resuspended in fluorescien isothiocyanate (FITC)-caspase substrateand the antibodies were added at concentrations of 5 ug/ml and 25 ug/ml(not shown), separately. The assay was incubated for 2 hours at 37° C.After washing, the cells were analyzed by FACS. The CCR4IgG (R4IgG) andCCR4scFvFc (R4Fc) test antibodies, and irrelevant control 80RIgG and38BscFvFc antibodies were included in the assay.

FIG. 11 are bar charts showing proliferation of mono- and co-cultures ofregulatory and effector T cells in presence of 1567scFv, 1567 cIgG1(panel A) and control (80R IgG1) antibodies (panel B). The CD4⁺ T cellswere sorted into CD4⁺/CD25^(high) (Regulatory) and CD4⁺/CD25⁻ (Effector)T cells by FACS. The Tregs and effector T cells were cultured at adensity of 2500 cells/well either alone or as a 1:1 co-culture with25,000 irradiated (3000 rad) CD3-depleted PBMCs used as antigenpresenting cells (APC). The cultures were stimulated by 0.05 μg/mlperipheral blood anti-CD3 and 1 μg/ml soluble anti-CD28. Proliferationwas measured on day 5 by ³H-labelled thymidine incorporation using ascintillation counter. The percent proliferation was normalized toCD4⁺/CD25⁻ T-effector cells without antibody treatment.

FIG. 12 are schematic of FACS scans showing binding of anti-human CCR4(1567), chimeric IgG1, and 80R (control IgG1) to human (panel A) andmacaque CD4⁺ T Cells (panel B). CD4⁺ T cells were incubated with 1567cIgG or 80R IgG and then labeled with FITC-anti-human Fc specificsecondary IgG. Cells were later stained for surface receptors CD4 andCD25, followed by fixation, and permeabilization for intracellularstaining of FOXP3. Panels C and D enumerate binding of the respectivecell types depicted in panels A and B.

FIG. 13 are schematics showing FACs analysis illustrating theHumanization of Mab1567. The left panel shows FACS staining of theparental murine 1567 scFv-phage and the humanized Vh-mouse V1 hybridscFv-phage. The right panel shows the binding of 3 of the 22 hybridmouse Vh-human V1 scFv-phage that were isolated from the human VL chainshuffled library.

FIGS. 14A and 14B are a pair of graphs showing that upon comparing thebinding activity of h1567-scFvFc to CCR4+ cell Mac-1 with mouse 1567,1567mscFv-Fc, humanized 1567 still remained good binding activity toMac-1 cells though not as good as m1567.

FIG. 15 is a schematic drawing of hCCR4. Differences in amino acidsbetween human and mouse are black circles, differences between human andguinea pig are green circles, and differences between guinea pig andmouse are half black/green circles. Also shown are putative N and Oglycosylation sites and tyrosines “Y” that may undergopost-translational sulfation.

FIG. 16 is an illustration showing procedures for In Vivo TherapeuticAntibody Gene Transfer in Mice. Gene transfer is accomplished using anewly reported rAAV serotype 8 (AAV-8) vector.

FIG. 17 is a graph showing alanine-scanning analysis of functionalresidues in the CDRs of humanized 1567 for binding to human CCR4 on cellsurface.

DETAILED DESCRIPTION

The present invention provides humanized monoclonal antibodies specificagainst chemokine (C—C motif) receptor 4 (CCR4). More specifically, theinvention provides a therapeutic antibody that eliminates malignant CTCLcells while minimizing collateral damage to an already compromisedimmune system. The antibodies are respectively referred to herein ishuCCR4 antibodies.

Cutaneous T-cell lymphomas (CTCLs) are a heterogenous group oflymphoproliferative disorders causes by clonally derived skin homing Tcells. CTCL cells uniformly express CCR4. Specifically, CCR4 is aprominent feature of malignant T cells in MF, cutaneous ALCL, androughly 50% of nodal ALCL. Unlike CLA, it is reliably expressed inSezary syndrome and during large cell transformation of MF and is alsoexpressed by other T lymphoid malignancies that can involve skin, suchas Adult T Cell Leukemia/Lymphoma (ATLL). Expression of CCR4 is limitedamongst non-malignant cells and absent on neutrophils, monocytes, or Bcells. Importantly, CCR4 is absent on nave T cells, and present on fewerthan half of all memory T cells. The reliable expression of CCR4 on CTCLcells, and its limited expression on other immune cells, makes targetedtherapy of CCR4 an attractive goal for these malignancies.

While some progress has been made in identifying small moleculeinhibitors that are relatively selective for CCR4, specific monoclonalantibodies against CCR4 are an attractive target for immunotherapy ofCTCL because of their exquisite binding specificity. In addition, the invivo effector functions that are mediated through Fc binding to Fcγreceptors can be exploited to kill tumor cells. The precise propertiesof Mabs that are required for optimal in vivo immunodepleting activityare not known, but antibodies can be selected to act as either asreceptor agonists or antagonists, and/or to promote or inhibit receptordimerization and/or internalization. Different immune mechanisms ofantibody-mediated tumor clearance have also been identified. Forexample, Mab-mediated recruitment of natural killer cells to tumors canoccur through the Fc-γ activation of receptors on these immune effectorcells, a process known as antibody-dependent cellular cytoxicity (ADCC).Other immune mechanisms include complement dependent cytotoxcicity (CDC)and antibody dependent cellular phagocytosis (ADCP). Additionalmechanisms related to intrinsic Mab activities include: blockade ofligand binding or hetero-dimerization, inhibition of downstreamsignaling of Akt, and acceleration of receptor internalization. Thelatter mechanism is particularly effective because ligand-inducedendocytosis and degradation of active receptor tyrosine kinases (RTKs)is considered a major physiological process underlying attenuation ofgrowth-promoting signals.

Leukocyte trafficking, which is critically regulated by chemokines andtheir receptors, share many of the characteristics of tumor cellinfiltration and metastasis. While expression of the chemokine receptorCCR4 by tumor cells is associated with skin involvement, CCR4 also hasan important role in both normal and tumor immunity. In a subset of CTCLpatients with HTLV-1 associated Adult T-cell leukemia/lymphoma (ATLL),the tumor cells themselves function as regulatory T (Treg) cells,contributing to tumor survival in the face of host anti-tumor immuneresponses. In other types of cancers, the chemokines TARC/CCL17 andMDC/CCL22, specific ligands for CCR4 that are produced by tumor cellsand the tumor microenvironment, attract CCR4⁺ Treg cells to the tumor,where they create a favorable environment for tumor escape from hostimmune responses. Thus, a therapeutic anti-CCR4 Mab is the idealtreatment modality for many different cancers, not only to directly killthe CCR4⁺ tumor cells, but also to overcome the suppressive effect ofCCR4 Treg cells on the host immune response to tumor cells.

In one aspect the present invention provides a humanized monoclonalantibody that specifically binds CCR4 proteins. Binding of this antibodyto the CCR4 receptor, interrupts ligand or agonist binding of CCR4.Exemplary ligands or agonists that compete for binding to the CCR4, andwhich are blocked in the presence of the invented antibody, include, butare not limited to, CCL17, CCL22, and vMIP-III. By a variety ofmechanisms, the antibody decreases ligand-induced chemotaxis ofcutaneous T cell lymphoma cells (CTCL cells). The huCCR4 antibody ismonovalent or bivalent and comprise a single or double chain.Functionally, the binding affinity of the huCCR4 antibody is about 1nM⁻¹ or less. The sequence of the antibody is engineered from and thus,may comprises one or more antigen-binding regions of murine CCR4antibody 1567. The hCCR4 antibody binds the same epitope as murine CCR4Mab 1567. The glycosylation of the Fc region of the antibody is modifiedto alter CCR4 binding or CCR4 ligand-blocking characteristics. Forinstance, the fucose content of the Fc region is decreased compared towild type. Furthermore, the antibody comprises a therapeutic agentincluding, but not limited to, a toxin, a radiolabel, a siRNA, or acytokine.

The huCCR4 antibody is capable of inducing cell death. Cell death isinduced by either direct or indirect mechanisms. For example, the huCCr4antibody binds CCR4 expressed on the surface of the target cell andleads to the death of that CCR4-expressing cell via intracellularsignaling pathways. For instance, CCR4 binding by the huCCR4 antibodycan lead to complement-dependent cytotoxicity (CDC). Alternatively, thehuCCR4 antibody binds CCR4, and leads to the recruitment of a secondcell type that will kill the CCR4-expressing target cell. Exemplarymechanisms by which the huCCR4 antibody mediates cell death byrecruitment of a second cell type include, but are not limited to,antibody-dependent cellular toxicity (ADCC) and antibody dependentcellular phagocytosis (ADCP). Target CCR4-expressing cell types comprisetumor and regulatory, or supplementary, T cells (also referred to asTreg cells).

The heavy chain CDRs of the huCCR4 antibody have the followingsequences: CDRH1: GYTFASYY (SEQ ID NO:5); CDRH2: WINPGNVNTKYNEKFKG (SEQID NO:6); and CDRH3: STYYRPLDY (SEQ ID NO:7). The light chain CDRs ofthe huCCR4 antibody have the following sequences: CDRL1:KSSQSILYSSNQKNYLA (SEQ ID NO:8); CDRL2: WASTRES (SEQ ID NO:9); andCDRL3: HQYLSSYT (SEQ ID NO:10).

huCCR4 VH nucleotide sequence: (SEQ ID NO: 1)CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGAGCTTCCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCGCCAGCTACTACATGCACTGGATGCGGCAGGCACCTGGACAGGGCCTCGAATGGATCGGCTGGATCAACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGGCAGGGCCACCCTGACCGTGGACACCAGCACCAACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAGCACCTACTACCGGCCCCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGChuCCR4 VH amino acid sequence: (SEQ ID NO: 2)QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARST YYRPLDYWGQGTLVTVSShuCCR4 V_(L) nucleotide sequence: (SEQ ID NO: 3)GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCATCCTGTACAGCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCACCAGTACCTGAGCAGCTACACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG huCCR4 V_(L) amino acid sequence:(SEQ ID NO: 4) DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSS YTFGQGTKLEIK

In various embodiments, HCDR2 includes the amino acid sequence ofWINXXNXNXKYNEKFKG (SEQ ID NO:11), HCDR3 includes the amino acid sequenceof SXYXXPLDX (SEQ ID NO: 12), LCDR1 includes the amino acid sequence ofKSSQSXLYSXXXXNYLA (SEQ ID NO: 13); LCDR2 includes the amino acidsequence of WASXXES (SEQ ID NO: 14); and LCDR3 includes the amino acidsequence of HQYLXXYT (SEQ ID NO: 15). As used herein X is meant todenote any natural, unnatural amino acid or amino acid analogue. Forexample, X is an alanine.

Antibodies

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” is meant that the antibody reacts with one or moreantigenic determinants of the desired antigen and does not react withother polypeptides. Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and F_(ab) expressionlibraries.

A single chain Fv (“scFv”) polypeptide molecule is a covalently linkedV_(H):V_(L) heterodimer, which can be expressed from a gene fusionincluding V_(H)- and V_(L)-encoding genes linked by a peptide-encodinglinker. (See Huston et al. (1988) Proc Nat Acad Sci USA85(16):5879-5883). A number of methods have been described to discernchemical structures for converting the naturally aggregated, butchemically separated, light and heavy polypeptide chains from anantibody V region into an scFv molecule, which will fold into a threedimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405;and 4,946,778.

Very large naïve human scFv libraries have been and can be created tooffer a large source of rearranged antibody genes against a plethora oftarget molecules. Smaller libraries can be constructed from individualswith infectious diseases in order to isolate disease-specificantibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43(1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)).

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain. The term“antigen-binding site,” or “binding portion” refers to the part of theimmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”CDRs for the VH and VL regions of the scFv antibodies are shown in FIG.2.

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. For example, antibodies maybe raised against N-terminal or C-terminal peptides of a polypeptide.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to a CCR4 epitope when the equilibrium bindingconstant (K_(d)) is ≤1 μM, preferably ≤100 nM, more preferably ≤10 nM,and most preferably ≤100 pM to about 1 pM, as measured by assays such asradioligand binding assays or similar assays known to those skilled inthe art.

An CCR4 protein of the invention, or a derivative, fragment, analog,homolog or ortholog thereof, may be utilized as an immunogen in thegeneration of antibodies that immunospecifically bind these proteincomponents.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the inventionby ascertaining whether the former prevents the latter from binding toCCR4. If the human monoclonal antibody being tested competes with thehuman monoclonal antibody of the invention, as shown by a decrease inbinding by the human monoclonal antibody of the invention, then it islikely that the two monoclonal antibodies bind to the same, or to aclosely related, epitope.

Another way to determine whether a human monoclonal antibody has thespecificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with theCCR4 protein, with which it is normally reactive, and then add the humanmonoclonal antibody being tested to determine if the human monoclonalantibody being tested is inhibited in its ability to bind CCR4. If thehuman monoclonal antibody being tested is inhibited then, in alllikelihood, it has the same, or functionally equivalent, epitopicspecificity as the monoclonal antibody of the invention. Screening ofhuman monoclonal antibodies of the invention, can be also carried out byutilizing CCR4 and determining whether the test monoclonal antibody isable to neutralize CCR4.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference).

Antibodies can be purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The term “monoclonal antibody” or “MAb” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanizedantibodies”, “human antibodies”, or “fully human antibodies” herein.Human monoclonal antibodies can be prepared by using trioma technique;the human B-cell hybridoma technique (see Kozbor, et al., 1983 ImmunolToday 4: 72); and the EBV hybridoma technique to produce humanmonoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized and may be produced by using human hybridomas(see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv (scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofCCR4 in a sample. The antibody can also be used to try to bind to anddisrupt a CCR4 activity.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively,an antibody can be engineered that has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. (See Stevenson etal., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987)). Preferred linkers aredescribed in the literature. (See, for example, Ramakrishnan, S. et al.,Cancer Res. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against CCR4

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Antibodies directed against a CCR4 protein (or a fragment thereof) maybe used in methods known within the art relating to the localizationand/or quantitation of a CCR4 protein (e.g., for use in measuring levelsof the CCR4 protein within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies specific to a CCR4 protein, or derivative,fragment, analog or homolog thereof, that contain the antibody derivedantigen binding domain, are utilized as pharmacologically activecompounds (referred to hereinafter as “Therapeutics”).

An antibody specific for a CCR4 protein of the invention can be used toisolate a CCR4 polypeptide by standard techniques, such asimmunoaffinity, chromatography or immunoprecipitation. Antibodiesdirected against a CCR4 protein (or a fragment thereof) can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may used as therapeutic agents. Such agentswill generally be employed to treat or prevent cancer in a subject,increase vaccine efficiency or augment a natural immune response. Anantibody preparation, preferably one having high specificity and highaffinity for its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withan activity of the CCR4 protein.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies specifically binding a CCR4 protein or a fragment thereof ofthe invention, as well as other molecules identified by the screeningassays disclosed herein, can be administered for the treatment of canceror other proliferative disorders in the form of pharmaceuticalcompositions. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington: The Science And Practice OfPharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co.,Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities,Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa.,1994; and Peptide And Protein Drug Delivery (Advances In ParenteralSciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

An antibody according to the invention can be used as an agent fordetecting the presence of CCR4 (or a protein or a protein fragmentthereof) in a sample. Preferably, the antibody contains a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., F_(ab), scFv, orF_((ab)2)) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Pharmaceutical Compositions

The antibodies or agents of the invention (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the antibody oragent and a pharmaceutically acceptable carrier. As used herein, theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with an CCR4 activity. Alsoprovided are methods of identifying compounds useful to treat cancer.The invention also encompasses compounds identified using the screeningassays described herein.

For example, the invention provides assays for screening candidate ortest compounds which modulate the CCR4 carbonic anhydrase activity. Thetest compounds of the invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. (See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates an CCR4activity.

In another embodiment, at least one CCR4 protein is provided, which isexposed to at least one neutralizing monoclonal antibody. Formation ofan antibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat cancer or aproliferative disease or disorder, particularly a renal proliferativedisorder.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a CCR4 neutralizingantibody. Additionally, the antigen may be a CCR4 protein, or a portionthereof (e.g., the CA domain).

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. In the case of cell-free assayscomprising the membrane-bound forms of the CCR4 proteins, it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the proteins are maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody or the antigen (e.g. the CCR4 protein or the CA domain thereof)can be immobilized utilizing conjugation of biotin and streptavidin.Biotinylated antibody or antigen molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well-known withinthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, other antibodies reactive with the antibody orantigen of interest, but which do not interfere with the formation ofthe antibody-antigen complex of interest, can be derivatized to thewells of the plate, and unbound antibody or antigen trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using such other antibodiesreactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic Assays

Antibodies of the present invention can be detected by appropriateassays, e.g., conventional types of immunoassays. For example, asandwich assay can be performed in which a CCR4 protein or fragmentthereof (e.g., the CA domain) is affixed to a solid phase. Incubation ismaintained for a sufficient period of time to allow the antibody in thesample to bind to the immobilized polypeptide on the solid phase. Afterthis first incubation, the solid phase is separated from the sample. Thesolid phase is washed to remove unbound materials and interferingsubstances such as non-specific proteins which may also be present inthe sample. The solid phase containing the antibody of interest bound tothe immobilized polypeptide is subsequently incubated with a second,labeled antibody or antibody bound to a coupling agent such as biotin oravidin. This second antibody may be another anti-CCR4 antibody oranother antibody. Labels for antibodies are well-known in the art andinclude radionuclides, enzymes (e.g. maleate dehydrogenase, horseradishperoxidase, glucose oxidase, catalase), fluors (fluoresceinisothiocyanate, rhodamine, phycocyanin, fluorescarmine), biotin, and thelike. The labeled antibodies are incubated with the solid and the labelbound to the solid phase is measured. These and other immunoassays canbe easily performed by those of ordinary skill in the art.

The anti-CCR4 antibodies and scFv antibodies of the invention, whenjoined to a detectable moiety, provides a way for detecting “canceroustissue” or tissue subject to aberrant cell proliferation and thereforeat risk for cancer. In addition to tissue that becomes cancerous due toan in situ neoplasm, for example, the antibody-detectable moietyconjugates also provides a method of detecting cancerous metastatictissue present in distal organs and/or tissues. Thus such tissue may bedetected by contacting tissue suspected of being cancerous with theantibody-detectable moiety under appropriate conditions to cause thedetectable moiety to be detected in cancerous tissue, thereby detectingthe presence of cancerous tissue.

The detectable moieties can be conjugated directly to the antibodies orfragments, or indirectly by using, for example, a fluorescent secondaryantibody. Direct conjugation can be accomplished by standard chemicalcoupling of, for example, a fluorophore to the antibody or antibodyfragment, or through genetic engineering. Chimeras, or fusion proteinscan be constructed which contain an antibody or antibody fragmentcoupled to a fluorescent or bioluminescent protein. For example,Casadei, et al., describe a method of making a vector construct capableof expressing a fusion protein of aequorin and an antibody gene inmammalian cells.

As used herein, the term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject (such as a biopsy), as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect cancer, a cancer cell, or a cancer-associated cell (suchas a stromal cell associated with a tumor or cancer cell) in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of CCR4 include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. Furthermore, in vivo techniques for detection ofCCR4 include introducing into a subject a labeled anti-CCR4 antibody.For example, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. In embodiments, the invention provides a non-invasive methodof detecting a tumor or cancer cell in a subject. The subject isadministered an antibody or scFv antibody of the invention, where theantibody is linked to a detectable moiety (i.e., any moiety capable ofbeing detected by, e.g., fluorescent, chemical, chemiluminescent,radioactive, or other means known in the art), the antibody is allowedto localize to the tumor then is detected by observation of thedetectable moiety.

In the case of “targeted” conjugates, that is, conjugates which containa targeting moiety—a molecule or feature designed to localize theconjugate within a subject or animal at a particular site or sites,localization refers to a state when an equilibrium between bound,“localized”, and unbound, “free” entities within a subject has beenessentially achieved. The rate at which such an equilibrium is achieveddepends upon the route of administration. For example, a conjugateadministered by intravenous injection to localize thrombi may achievelocalization, or accumulation at the thrombi, within minutes ofinjection. On the other hand, a conjugate administered orally tolocalize an infection in the intestine may take hours to achievelocalization. Alternatively, localization may simply refer to thelocation of the entity within the subject or animal at selected timeperiods after the entity is administered. By way of another example,localization is achieved when an moiety becomes distributed followingadministration.

In all of the above cases, a reasonable estimate of the time to achievelocalization may be made by one skilled in the art. Furthermore, thestate of localization as a function of time may be followed by imagingthe detectable moiety (e.g., a light-emitting conjugate) according tothe methods of the invention, such as with a photodetector device. The“photodetector device” used should have a high enough sensitivity toenable the imaging of faint light from within a mammal in a reasonableamount of time, and to use the signal from such a device to construct animage.

In cases where it is possible to use light-generating moieties which areextremely bright, and/or to detect light-generating fusion proteinslocalized near the surface of the subject or animal being imaged, a pairof “night-vision” goggles or a standard high-sensitivity video camera,such as a Silicon Intensified Tube (SIT) camera (e.g., from HammamatsuPhotonic Systems, Bridgewater, N.J.), may be used. More typically,however, a more sensitive method of light detection is required.

In extremely low light levels the photon flux per unit area becomes solow that the scene being imaged no longer appears continuous. Instead,it is represented by individual photons which are both temporally andspatially distinct form one another. Viewed on a monitor, such an imageappears as scintillating points of light, each representing a singledetected photon. By accumulating these detected photons in a digitalimage processor over time, an image can be acquired and constructed. Incontrast to conventional cameras where the signal at each image point isassigned an intensity value, in photon counting imaging the amplitude ofthe signal carries no significance. The objective is to simply detectthe presence of a signal (photon) and to count the occurrence of thesignal with respect to its position over time.

At least two types of photodetector devices, described below, can detectindividual photons and generate a signal which can be analyzed by animage processor. Reduced-Noise Photodetection devices achievesensitivity by reducing the background noise in the photon detector, asopposed to amplifying the photon signal. Noise is reduced primarily bycooling the detector array. The devices include charge coupled device(CCD) cameras referred to as “backthinned”, cooled CCD cameras. In themore sensitive instruments, the cooling is achieved using, for example,liquid nitrogen, which brings the temperature of the CCD array toapproximately −120° C. “Backthinned” refers to an ultra-thin backplatethat reduces the path length that a photon follows to be detected,thereby increasing the quantum efficiency. A particularly sensitivebackthinned cryogenic CCD camera is the “TECH 512”, a series 200 cameraavailable from Photometrics, Ltd. (Tucson, Ariz.).

“Photon amplification devices” amplify photons before they hit thedetection screen. This class includes CCD cameras with intensifiers,such as microchannel intensifiers. A microchannel intensifier typicallycontains a metal array of channels perpendicular to and co-extensivewith the detection screen of the camera. The microchannel array isplaced between the sample, subject, or animal to be imaged, and thecamera. Most of the photons entering the channels of the array contact aside of a channel before exiting. A voltage applied across the arrayresults in the release of many electrons from each photon collision. Theelectrons from such a collision exit their channel of origin in a“shotgun” pattern, and are detected by the camera.

Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification. An exemplary microchannel intensifier-based single-photondetection device is the C2400 series, available from Hamamatsu.

Image processors process signals generated by photodetector deviceswhich count photons in order to construct an image which can be, forexample, displayed on a monitor or printed on a video printer. Suchimage processors are typically sold as part of systems which include thesensitive photon-counting cameras described above, and accordingly, areavailable from the same sources. The image processors are usuallyconnected to a personal computer, such as an IBM-compatible PC or anApple Macintosh (Apple Computer, Cupertino, Calif.), which may or maynot be included as part of a purchased imaging system. Once the imagesare in the form of digital files, they can be manipulated by a varietyof image processing programs (such as “ADOBE PHOTOSHOP”, Adobe Systems,Adobe Systems, Mt. View, Calif.) and printed.

In one embodiment, the biological sample contains protein molecules fromthe test subject. One preferred biological sample is a peripheral bloodleukocyte sample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence of CCR4or a CCR4-expressing cell in a biological sample. For example, the kitcan comprise: a labeled compound or agent capable of detecting a canceror tumor cell (e.g., an anti-CCR4 scFv or monoclonal antibody) in abiological sample; means for determining the amount of CCR4 in thesample; and means for comparing the amount of CCR4 in the sample with astandard. The standard is, in some embodiments, a non-cancer cell orcell extract thereof. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect cancer in a sample.

Methods of Treatment

The invention provides for both prophylactic and therapeutic methods oftreating a subject at risk of (or susceptible to) a cancer, or othercell proliferation-related diseases or disorders. Such diseases ordisorders include but are not limited to, e.g., those diseases ordisorders associated with aberrant expression of CCR4. For example, themethods are used to treat, prevent or alleviate a symptom of ahematologic cancer such cutaneous T-cell Lymphoma (CTCL), mycosisfungoides (MF), primary cutaneous anaplastic large cell Lymphoma(cutaneous ALCL), Sezary syndrome, or adult T cell Leukemia/Lymphoma(ATLL). Alternatively, the methods are used to treat, prevent oralleviate a symptom of a solid tumor such as s breast cancer, lungcancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer,brain cancer, liver cancer, pancreatic cancer or stomach cancer.

Accordingly, one aspect, the invention provides methods for preventing,treating or alleviating a symptom cancer or a cell proliferative diseaseor disorder in a subject by administering to the subject a monoclonalantibody or scFv antibody of the invention. For example, a huCCR4antibody may be administered in therapeutically effective amounts.

Subjects at risk for cancer or cell proliferation-related diseases ordisorders include patients who have a family history of cancer or asubject exposed to a known or suspected cancer-causing agent.Administration of a prophylactic agent can occur prior to themanifestation of cancer such that the disease is prevented or,alternatively, delayed in its progression.

In another aspect, tumor cell growth is inhibited or suppressor T-cellactivity is decreased by contacting a cell with an CCR4 antibody of theinvention. The cell is any cell that express CCR4. For example the cellis T-cell.

Also included in the invention are methods of increasing or enhancing animmune response to an antigen. An immune response is increased orenhanced by administering to the subject a monoclonal antibody or scFvantibody of the invention. The antigen is a viral (e.g. HIV), bacterial,fungal or tumor antigen. The immune response is a natural immuneresponse. By natural immune response is meant an immune response that isa result of an infection. The infection is a chronic infection.

Alternatively, the immune response is a response induced due to avaccination. Accordingly, in another aspect the invention provides amethod of increasing vaccine efficiency by administering to the subjecta monoclonal antibody or scFv antibody of the invention and a vaccine.The antibody and the vaccine are administered sequentially orconcurrently. The vaccine is a tumor vaccine a bacterial vaccine or aviral vaccine.

The immune response is augmented for example by augmenting antigenspecific T effector function.

Combinatory Methods

The invention provides treating cancer in a patient by administering twoantibodies that bind to the same epitope of the CCR4 protein or,alternatively, two different epitopes of the CCR4 protein. Also, thecancer is treated by administering a first antibody that binds to CCR4and a second antibody that binds to a protein other than CCR4.

Additionally, the invention provides administration of an antibody thatbinds to the CCR4 protein and an anti-neoplastic agent, such a smallmolecule, a growth factor, a cytokine or other therapeutics includingbiomolecules such as peptides, peptidomimetics, peptoids,polynucleotides, lipid-derived mediators, small biogenic amines,hormones, neuropeptides, and proteases. Small molecules include, but arenot limited to, inorganic molecules and small organic molecules.Suitable growth factors or cytokines include an IL-2, GM-CSF, IL-12, andTNF-alpha. Small molecule libraries are known in the art. (See, Lam,Anticancer Drug Des., 12:145, 1997.)

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: General Methods

Acquisition, Characterization, and Quantification of CCR4-Bearing CellTypes.

Fresh, high-quality buffy-coats of human white bloods cells are receiveddaily from the Blood Banks at Children's Hospital, Dana-Farber CancerCenter and Brigham and Women's Hospital. Upon receiving these samples,white blood cell populations are fractionated into granulocyte and PBMCsubunits using Ficoll gradients.

Reagents are also tested on peripheral blood from leukemic CTCLpatients, as a “reality check” to ensure that experimental data, withrespect to normal CCR4-bearing populations, is applicable to true targetpopulations. Such samples are available by informed consent fromleukemic CTCL patients. PBMC is prepared as follows. Human granulocyteor PBMC is incubated on ice with Mab1567 for 45 minutes. Equal numbersof cells are treated in parallel with control human IgG1 Mabs or withPBS. “Charged” Dynabeads are added to the MAb-bound cells and incubatedwith gentle rotation. Following this incubation, Mab-bound and non-boundpopulations are magnetically separated. These steps generate 3 cellpopulations: 1) cells depleted via a “mock” antibody (PBS); 2) cellsdepleted via a “negative control” or “irrelevant” Mab; and 3) cellsdepleted via Mab 1567. The three types of cell populations areextensively immunophenotyped for comparison with each other using atleast 10 different fluorescent antibody cocktails.

The relative proportions of various lymphocyte populations among the 3different cell populations are immunophenotyped, by using a 6-colorCytomation cytometer and an extensive repertoire offluorescently-labeled Mabs, more than 50 non-overlapping subsets oflymphocytes within human peripheral blood are distinguished (Campbell J.J. et al. Nature 1999, 400:776-780; Campbell J. J. et al. J Immunol2001, 166:877-884; Soler D. et al. Blood 2003, 101:1677-1682; Kunkel E.J. et al. Am J Pathol 2002, 160:347-355; Campbell J. J. et al. J CellBiol 1998, 141:1053-1059). Memory, effector and naïve CD4 T cellsubsets; memory, effector and naïve CD8 T cell subsets; memory and naïveB cells subsets; NK subsets; NKT subsets; Treg; and dendritic cellsubsets are identified and quantified using this method. In thegranulocyte populations, the relative proportions of eosinophils,basophils and neutrophils are quantified.

Quantification of CCR4 Expression Levels.

FACS analysis is used to determine the number of CCR4 molecules on thesurface of CD4⁺/CCR4⁺ T cells by comparing relative levels of FACSbinding of the Mab 1567 at under physiological and saturated conditions.Cells from different healthy individuals are examined to evaluateheterogeneity of expression patterns in the population, as well as fromthe blood of patients with Szary syndrome, and other CTCLs. AMann-Whitney U test is used to determine if any significant differencesexist between CCR4 level of expression across various cell types. Thesimultaneous analysis of multiple markers from the same donor allows fora paired t-test with a two-tailed distribution analysis to be applied tothe analysis of CD4⁺ T cell subsets.

Immunohistochemical Analyses

Frozen CTCL tissue blocks are retrieved from the SPORE BiospecimenAccess and Analysis Core. Frozen tissue is also obtained from normalskin samples from plastic surgery cases. Following fixation, sequentialsections are incubated with Mab1567 (Mab 1G1 serves as a positivecontrol), washed, and then incubated with horseradish peroxidase(HRP)-labeled anti-human IgG1 Mab. Antibody localization will bevisualized using a peroxidase reaction using diaminobenzidine (DAB) as achromogen (Dako). Slides are counterstained with methylene green,dehydrated, and mounted with Polymount (Polysciences). A control slidesubstituting an irrelevant human IgG1 Mab for Mab 1567 is included inall cases.

Example 2: Distinguishing CTCL Clones and Normal T Cells in LeukemicCTCL Blood

Immunophenotypic diversity observed within CTCL blood samples resultsfrom the presence of persisting “normal” CD4 T cells in the midst of thetransformed CTCL cells. To identify leukemic CTCL clonally-expandedpopulations amongst the healthy cells, a systematic method ofcharacterization of leukemic cells was developed based on differentialexpression of unique T cell receptors (TCRs). Individual T cell cloneseach express a unique TCR heterodimer consisting of an α-chain and aβ-chain. Each β-chain is derived from random recombination of “V”, “D”and “J” segments within the TCR locus of the T cell genome duringdevelopment (Abbas A, Lichtman, A H. Cellular And Molecular Immunology,Updated Edition (ed 5). Elsevier Saunders 2005). There are only alimited number of TCR-Vβ□ genes available for recombination. The “V”portion of TCRs on the surface of an individual T cell can be recognizedby monoclonal antibodies via flow cytometry. Such antibodies arecommercially available for a large proportion of the known TCR-Vβs.Approximately half of the leukemic CTCL patients who were testedpossessed a grossly overrepresented population of CD4 T cells bearing asingle TCR-Vβ. These expanded populations (never seen in normal donors)correspond to the clonally-derived, transformed CTCL component of theCD4 pool. Expanded populations that express TCR-V-β segments not coveredby the antibody panel likely account for the leukemic CTCL patientswithout an identifiable clone. Once an expanded CD4 T clone from aparticular leukemic CTCL patient was identified, the resulting uniqueprofile was used to discriminate CTCL cells from the remaining “normal”CD4 T cells for that particular patient. Multi-color flow cytometry wasthen used to sequester cells displaying this unique profile as well asextensively analyze the phenotype of these pure CTCL cells.

Example 3: CCR4 is a Marker for CTCL Clones

Our analysis of CTCL clones was primarily borne from the hypothesis thatCTCL cells are transformed versions of normal skin-homing memory CD4 Tcells. Cutaneous CD4 lymphocytes from normal human blood have beenidentified by their co-expression of the cutaneous lymphocyte antigen(CLA) (Campbell J. J. et al. J Cell Biol 1998, 141:1053-1059) and thechemokine receptor CCR4 (Campbell J. J. et al. Nature 1999, 400:776-780;Kunkel E. J. et al. Am J Pathol 2002, 160:347-355). In addition, thechemokine receptor CCR10 is expressed exclusively by a subset ofcutaneous CD4 T cells (Soler D. et al. Blood 2003, 101:1677-1682). Theexpression of CLA, CCR4 and CCR10, identified by clonal expression ofunique TCR-V-β segments, on CTCL cells was analyzed (FIG. 1). CTCL cellsexpressed each of these markers to varying degrees. This finding can beexplained by each of the following two hypotheses: 1) CTCL cells havederived from a non-cutaneous CD4 population that has usurped thecutaneous homing markers expressed by normal skin T cells; or 2) CTCLcells are transformed cells derived from normal cutaneous-homing Tcells. To distinguish between these two potential sources ofCLA⁺/CCR4⁺/CCR10⁺ clones, CTCL cells purified from skin lesions andperipheral blood sampled from leukemic CTCL patients were examined (FIG.2). The results showed that CCR4 was expressed by essentially all CTCLcells from both sources. Most of the CTCL cells from the skin lesionsexpressed CLA, in marked contrast to CTCL cells from blood, whichexpressed significantly reduced levels of CLA. This finding suggestedthat loss of CLA expression correlated with increasing numbers of CTCLcells in patient blood. Because CLA is an adhesion molecule necessaryfor the homing of cutaneous T cells to the skin it is reasonable toposit that loss of CLA expression (and therefore exclusion from theskin) plays an important role in the extracutaneous spread (e.g., blood,lymph nodes, viscera) of more aggressive variants of CTCL. However, someforms of CTCL emerge and spread by a different pattern. CTCL of themycosis fungoides variant is often an indolent lymphoma, withtransformed cells occurring only rarely in the blood (Yawalkar N. et al.Blood 2003, 102:4059-4066; Robert C, Kupper T S. N Engl J Med 1999,341:1817-1828). In some cases, the disease advances into leukemic CTCL,with large numbers of cells appearing in the blood. In other cases, CTCLfirst presents as a leukemic disease involving both blood and skin.

Data from previous studies have shown that CCL17 and CCL21 (both ligandsfor CCR4) are strongly expressed in lesional skin of CTCL patients(Ferenczi K. et al. J Invest Dermatol 2002, 119:1405-1410), facilitatingthe entry of and maintenance within skin of these malignant cells.However, without CLA to initiate the tethering of CTCL cells tocutaneous post-capillary venules, CCR4 cannot be engaged by its ligandswithin skin and the CTCL cells remain in the blood.

Example 4: Validation of CCR4 Flow Cytometry Studies

Before embarking on a project to target CTCL via CCR4, it was necessaryto validate that the staining patterns in FIGS. 1-2 accurately representCCR4 expression by CTCL cells. Fortunately, because CCR4 is achemotactic receptor, CCR4 function can be readily assessed bywell-established in vitro migration assays. Peripheral blood wasobtained from 3 different leukemic CTCL patients bearing apreviously-identified TCR-V-β clonal expansion. For each CTCL patient,peripheral blood mononuclear cells (PBMCs) from a normal donor wereassessed in parallel as a positive control. Migratory ability wasmeasured via the widely-used in vitro transwell chemotaxis assay (KunkelE. J. et al. Am J Pathol 2002, 160:347-355; Campbell J. J. et al. J CellBiol 1996, 134:255-266; Warnock R. A. et al. J Exp Med 2000, 191:77-88)(FIG. 3).

Cells were placed in an optimized gradient of a CCR4 ligand, a CCR7ligand, or in the absence of chemokine. CTCL cells were stained for flowcytometry with CD4 and the appropriate anti-TCRVβ. Normal PBMCs werestained with CD4, CLA and CD45RA to identify naïve CD4 cells(CD4⁺/CD45RA^(high)) and cutaneous memory CD4 cells(CD4⁺/CD45RA^(lo/neg)/CLA⁺). All 3 cell types responded well to CCR7ligand, but naïve cells responded best to this chemokine. Naïve CD4cells did not respond detectably to CCR4 ligand. Both CTCL cells andnormal cutaneous memory cells responded well to CCR4 ligand and, indeed,CTCL cells responded better. Thus, this independent functional assayproves that CTCL cells are responsive to CCR4 ligand.

T cells were also isolated from skin lesions of CTCL patients usingshort term explant cultures (Clark R. A. et al. J Invest Dermatol 2006,126:1059-1070). Following analysis by flow cytometry, it was determinedthat CTCL skin lesions from all stages of disease contained a uniquepopulation of T cells with higher forward and side scatter. Thesehigh-scatter T cells were not seen in either normal skin or skin lesionsof psoriasis, or in contact dermatitis or atopic dermatitis, suggestingthat they do not represent antigen-activated T cells. The high-scatter Tcell population was also evident in the blood of later-staged CTCL withknown blood involvement but was absent in earlier stages of the disease.In late-stage patients, where malignant T cells were identified by theirparticular TCR Vβ expression, the high scatter T cell population fromboth skin and blood was clonal and malignant (FIG. 4). All malignant Tcells from both blood and skin expressed CCR4, whereas the expression ofCLA within skin-derived cells was more variable. This ability todiscriminate malignant from benign T cells in early CTCL lesions enablesmore detailed study of the malignant T cells in this disorder and theirselective depletion by Mab1567 based immunotherapy.

Example 5: ALCL Lines as In Vitro and In Vivo Models of CTCL Cells

It is well-established that CTCL cells are difficult to perpetuate inculture or grow in immunodeficient mice. To circumvent this obstacle,several human skin-tropic ALCL cell lines that are robust in culture andin animal models are used (Kleinhans M et. al. Blood 2003,101:1487-1493; Li S. et al. Am J Pathol 2001, 158:1231-1237; Pfeifer W.et al. Am J Pathol 1999, 155:1353-1359; Zhang Q. et al. Proc Natl AcadSci USA 1996, 93:9148-9153). Moreover, these cells form subcutaneoustumors when injected into immunodeficient mice (Pfeifer W. et al. Am JPathol 1999, 155:1353-1359). Thus, these cells, called Mac-1 and Mac-2b,were immunophenotyped to determine if their expression profile ofskin-related chemokine receptors is comparable to that of CTCL cells.The results showed that like CTCL cells, these ALCL lines expressed highlevels of CCR4, CCR7 and CXCR4. These cell lines did not expresssignificant levels of any of the other known chemokine receptors,including, for example, CCR5. The Mac-1/2b cell lines serve asparticularly good models of CTCL because they are of human origin,express high levels of CCR4, and grow in immunodeficient mice. Theability of CCR4 Mabs to clear CCR4⁺ cells from a living animal isdirectly tested using an animal model of CTCL, created by injection ofthe Mac-1/2b cell line into immunodeficient mice. Because this modelemploys a cell line derived from human tissue, this method of Mabvalidation is superior to any other method using mouse cells. Theresults of these studies directly translate to human therapeuticstrategies.

Example 6: In Vivo Immunodepletion of CCR4-Expressing Human Skin-TropicALCL Tumor Cells by Mab1567

The IgG2b murine Mab1567 and control murine Mab (1D4) were used toestablish an immunotherapy model in severe combined immunodeficiency(SCID)/beige mice. There were 6 mice in each treatment group. Eachanimal received an intradermal (id.) injection of 1×10⁷ Mac-1 cells onone flank, followed immediately by intraperitoneal (i.p.) injection of 3mg Mab, followed by bi-weekly Mab injections. As shown in FIG. 5, therewas a marked inhibition of tumor cell growth. This is quite remarkableconsidering that these mice have no NK activity or ADCC, and that theantibody isotype IgG2b binds more avidly to immunoinhibitory FcγIIIreceptors than to immuno activating FcγIII receptors (Nimmerjahn F,Ravetch J V. Science 2005, 310:1510-1512). Studies performed with adifferent chimeric mouse Mab showed that in vivo anti-tumor activitycould be mediated indirectly by enhancement of phagocytic activitymediated by monocytes/macrophages (Ishida T, Ueda R. Cancer Sci 2006,97:1139-1146).

Example 7: Cloning of the Mouse Anti-CCR4 Mab1567

The murine anti-human CCR4 single-chain antibody (1567) was constructedfrom the hybridoma cell clone 205410 (provided by R&D systems Inc.)using known techniques. As shown in FIG. 6, 1567scFv binds specificallyto Mac-1 cells as well as stable 293T-CCR4 cells but not to parentalcontrol 293T cells. Moreover there is a 2-4 fold increase in apparentaffinity in converting the scFv to the soluble bivalent scFvFc(scFv-Hinge-CH2-CH3 of human IgG1).

Example 8: Expression of CCR4-Nt-Fc Fusion Protein for Epitope Mappingof 1567 Mab

A fusion protein was constructed to join a soluble version of theNH-terminus of CCR4 with a human IgG1 Fc domain. To show that thisfusion protein could be processed by a cell properly and undergo propersignal peptide cleavage, 293T cells expressing the F105-L3-Nt-hCCR4-Fcfusion protein were labeled with [³⁵S]-cysteine and [³⁵S]-methionine, or[³⁵S]-sulfate. Culture supernatants containing secreted proteins wereimmunoprecipitated with protein A sepharose beads and applied to aSDS-PAGE reducing gel for analysis. The results indicated that theCCR4-Nt-Fc fusion protein had been secreted from transfected 293T cellsfollowing proper signal peptide cleavage. The wild type CCR5-Nt-Fc, usedas a positive control, and the constructed CCR4-Nt-Fc proteins were alsoproperly modified by post-translational processing demonstrated bysulfation of the tyrosine residues (Y16, Y19, Y20 and Y22) in theN-terminal (Nt) regions. However, the mutant CCR5 protein, used here asa negative control, in which 4 Nt tyrosine residues have been mutated toaspartic acid (DDDD), was not sulfated. The CCR4-Nt-Fc fusion protein isabout 110 kDa and dimeric (bivalent) (FIG. 7).

The CCR4-Nt-Fc fusion proteins were also used to map the epitoperecognized by 1567mscFv-Fc. ELISA experiments showed that the 1567 Mabspecifically retained the CCR4-Nt-Fc protein, but did not bind theCCR5-Nt-Fc, an unrelated scFvFc (PS11) protein, or BSA. Thus, these datashow that the Mab1567 specifically recognizes the N-terminus of CCR4,however, the possibility remains that the epitope encompasses multipledomains, one of which is the N-terminal region.

Example 9: Inhibition of CCL22-Mediated Chemotaxis by m1567 Mab and 1567mscFv-Fc

The 1567 Mab and 1567 mscFv-Fc antibody were tested to assess theirability to inhibit CCL22-mediated chemotaxis of human ATCL tumor cells,Mac-1 cells. CCL22 is a well-characterized ligand for CCR4 that isexpressed on T-cells. Mab1567 (10 ng/mL & 20 ng/mL), 1567 mscFv-Fc or anIgG2b isotype control were placed in the upper chamber of a 12-welltranswell plate (of 5 μm pore size) in the presence of CCL22 (100 nM) in600 μL media housed in the lower chamber at 37° C. for 4 hours. Cellmigration into the lower chamber was determined by a FACS cell countusing Flow-Check Fluorospheres as reference (experiments performed induplicate). The migration assay results showed that both Mab1567 and1567 mscFv-Fc inhibited chemotaxis of human Mac-1 tumor cells at verylow antibody concentrations (ng/ml). Expectedly, the negative controlisotype Mab had no effect.

Example 10: 1567 mscFv-Fc-Mediated ADCC Activity

An ADCC assay was performed using the DELFIA EuTDA cytotoxicity kit.Normal, healthy, donor blood was obtained from Children's Hospital BloodBank and peripheral blood mononuclear cells (PBMCs) were isolated viaFicoll (two different human PBMC donors were tested, only one of whichis shown). Briefly, the target cell line, Mac-1, was labeled withfluorescent ligand BATDA reagent. Labeled Mac-1 cells (at a density of11×10⁴/well) were loaded into a 96-well plate in triplicates (FIG. 9).Different ratios (50:1, 25:1, and 12.5:1) of effector (PBMCs) to targetcells (labeled Mac-1 cells) were established within each set oftriplicate wells. Anti-CCR4 1567 mscFv-Fc (“R4”, in figure) or anirrevelant anti-HA 38B scFvFc (“38B”, in figure) were added to each wellat a concentration of 1 ug/ml or 5 ug/ml and incubated for 4 hours. Thespontaneous release control was established by culturing the labeledcells in the absence of any other factor while the maximum releasecontrol was established by adding lysis buffer to labeled cells.

The results showed that anti-CCR4 1567 mscFv-Fc induces significantADCC-mediated target cell death in all cases, however the effect wasmost extreme when the effector-to-target ratio was 50:1, halved in the25:1 case, and present in 12.5:1 case. Anti-CCR4 1567 mscFv-Fc appearedto be as effective at 1 ug/ml as it was at 5 ug/ml. Neither anti-HA3B3scFvFc nor the ab-free negative control displayed any ADCC activity.These studies demonstrate that the 1567 mscFv-Fc antibody mediatespotent ADCC activity via CCR4 expressed on human ATCL tumor cells (Mac-1cells).

Example 11: Construction and ADCC Activity of a Chimeric Human IgG1 Mab(cIgG)

The individual VH and VL genes from murine 1567 scFv were used toconstruct a chimeric human IgG1 Mab (cIgG). A repeat ADCC assay wasperformed in order to compare the activities of the 1567 mscFv-Fc and1567 cIgG. Although the background ADCC-mediated killing with theirrelevant 80R hIgG (anti-SARS antibody) or 38B scFvFc was high in thisexperiment, the 1567 (R4) cIgG and scFvFc treatments resulted in muchhigher ADCC activity (FIG. 10). Importantly, the ADCC activity of 1567cIgG was at least as potent that of 1567 mscFv-Fc

Example 12: 1567 cIgG Blocks Treg Activity

CD4⁺CD25^(high) (regulatory) and CD4⁺CD25⁻ (effector) T cells wereeither mono- or co-cultured (1:1 ratio, 2500 cells/well in bothconditions) with irradiated CD3-depleted PBMCs (to present antigen), andexposed to either 1567 cIgG or control 80R hIgG (anti-SARS Mab) addedinto the medium. Following exposure to antibody and stimulation byanti-CD3 or anti-CD28, T-cell cultures were sorted by FACS andproliferation was measured on day 5 by ³H-labelled thymidineincorporation using a scintillation counter. The percent proliferationwas normalized to CD4⁺CD25⁻ T effector cells without antibody treatment.

The results of this experiment showed that addition of the 1567 cIgGantibody augmented proliferation of CD4⁺/CD25⁺ (T effector cells (141%)compared to the proliferation of T effector cells without antibodytreatment (FIG. 11). Importantly, 1567 cIgG dramatically reversed thesuppression of T effectors by Tregs from 19.2% to 80% of theproliferation seen with untreated T effector cells. This abrogation ofsuppression was not seen with control 80R hIgG (anti-SARS Mab). Theseresults suggest that 1567 cIgG abrogates the suppressive activity ofTregs in co-cultures by acting on the CD4⁺/CD25⁻ effector T cells.

Example 13: 1567 cIgG Binds to Human and Rhesus Macaque CD4⁺ Teffectorand Treg Populations

FACS studies were performed on CD4 subpopulations from two human donorsand rhesus macaques each, only one representative donor of each is shownin FIG. 12. The results showed that the 1567 cIgG bound to 88% of humanCD4⁺CD25^(high)Foxp3⁺ cells with a phenotype consistent with Treg cells(see panel C of FIG. 12). A much lower percentage of 1567 cIgG bindingto the human CD4⁺CD25⁻ cells was seen, which is consistent with previousobservations that only a subset of CD4 memory T cells express CCD4.Similarly, CCR4 is expressed on 86% of CD4⁺CD25^(high)Foxp3⁺ rhesusmacaque cells (see panel D of FIG. 12). However, in the two macaquesthat were studied, CCR4 staining of the CD4⁺CD25⁻ cells, both FoxP3⁺ andFoxP3⁻ populations was higher than was observed with human cells. Theresults suggest that rhesus macaques can be used as a closeapproximation of the human condition to develop a CCR4 Mab therapy.

Example 14: Humanization of VH from Mab1567

Three-dimensional modeling of the murine VH region was performed. Adetailed all-atom model of the VH 1567 was constructed by usingSwissModeling Software. Briefly, the best matched sequences of 1567 withknown antibody structures in the Protein Data Bank (PDB) database wereselected and used to building a reliable three-dimensional model of thevariable regions. In parallel with modeling the structure, the aminoacid sequence of mouse 1567 and human VH domains were compared in theKabat database to find the most suitable human framework sequence forantibody humanization. A human VH framework sequence (McAb Ctm01,PDB:1ae6H) with 82% homology to the mouse sequence was selected as theframework template for humanization of 1567 VH. Two other considerationswere also taken into account during the VH humanization procedures: 1)key framework residues indicated by the three-dimensional model werekept and, 2) atypical amino acids that occur in less than 10-20% of thehuman VH genes were eliminated to avoid potential immunogenecity andwere replaced with a consensus amino acid residue from the same human VHsubgroup. A humanized VH gene (hVH) was designed and a human codonoptimized gene was synthesized. Finally, a hybrid hVH-mVL of 1567 scFvwas constructed and expressed as a phage-scFv to test its bindingactivity to the CCR4-expressing cell, Mac-1, by FACS (FIG. 13). Theresults of the FACS analysis showed that the hybrid hvh-mVL 1567(half-human/half mouse) retains at least 50% of the mouse 1567 bindingactivity.

Example 15: Humanization of VL from 1567 MAb

A similar strategy was used to humanize VL of 1567. A human Vkappaframework sequence (GenBank™ # ABG38372) with 84% homology to the mouse1567 VL was selected as framework template for humanization. Thus, ahumanized VH gene (hVH) and humanized VK gene (hVK) were designed andtheir codon optimized genes for expressing in human cells weresynthesized, and then hVL of 1567 was paired with hVH of 1567 to form afully humanized 1567 (567 hscFv). This scFv was further converted tosoluble bivalent scFvFc (1567hscFv-Fc). Binding activity of h1567-scFvFcto CCR4⁺ cell Mac-1 was tested and compared with mouse 1567,1567mscFv-Fc. As shown in FIGS. 14A and 14B, humanized 1567 stillremained good binding activity to Mac-1 cells though not as good asm1567.

Example 17: Humanized VH and Human Full VL Library Shuffle

Light chain swapping is a powerful antibody technique that is used forboth the humanization and augmentation of Mab binding affinity (SchierR. et al. J Mol Biol 1996, 255:28-43). It is possible that the humanizedVH is not perfectly modeled from the murine VH, potentially resulting insome loss of binding activity when murine VH is exchanged for thehumanized VH. Thus, a humanized VH-human VL shuffle library (1×10⁷member) was constructed to use in panning experiments with CCR4-PMPLs.Using this method, the library has a 1.2×10⁸ human Vkappa available forshuffling. Positive clones showing strong Mac-1 binding properties areevaluated as described in Example 15.

Example 18: Human VH Shuffle Library with Optimized hVL

A human VL with the best binding activity from the above example isidentified and used in combination with a human VH shuffle library tofurther enhance the binding affinity of the humanized antibody (Schier Ret al. J Mol Biol 1996, 255:28-43). This library is on the order of1×10⁸ members (a 3.5×10⁸ member human VH library is also available, seelink in Example 16). This sequential chain swapping methodology isstandard practice in the field and, importantly, the antibody thatemerges from this screen has the same epitope specificity as theparental Mab. Panning is performed using CCR4-PMPLs. Evaluation of thephage binding to >70% of Mac-1 cells is performed in a manner identicalto that described in Examples 15 and 16. A humanized 1567 Mab with anaffinity of ≤1 nM⁻¹, which is a benchmark standard that is used incommercial Mab development, is produced using this strategy. Thisbinding affinity allows hMabs 1567 to achieve therapeutically meaningfulserum concentrations when dosed at ≤10 mg/kg/dose in humans.

Example 19: Optimization of Mab Binding Affinity Using FocusedMutagenesis

The combined results of Examples 15-17 and DNA sequence analysis of theCDR regions provide a pattern of consensus amino acids that play asignificant role in binding activity. This information is used tofurther improve the binding affinity of the humanized 1567, if required,based on the cell binding and BiaCore™ binding studies (described inExample 21). Specifically, the pattern of conserved and variable aminoacids provides valuable information for selecting which amino acids inthe CDRs to subject to focused mutagenesis is concentrated on thevariable amino acids surrounding the conserved amino acids that areuncovered in the analysis (Schier R et al. Hum Antibodies Hybridomas1996, 7:97-105).

Example 20: Optimization of Mab Binding Affinity Using In Vitro AffinityMaturation

Higher affinity antibodies are obtained during the selection process bylowering the concentration of antigen in each subsequent round ofpanning (Marks J D. Methods Mol Biol 2004, 248:327-343). This modifiedpanning procedure is used when needed and as guided by the results ofthe BiaCore™ studies described herein. (Schier R et al. Hum AntibodiesHybridomas 1996, 7:97-105).

Example 21: Cell Binding Affinity Measurements of Anti-CCR4 scFvFcs andIgGs

Saturation binding studies on stable Cf2-CCR4⁺ cells are performed oneach of the purified 1567 scFvFc and IgG variants. The approximateaffinity of each antibody for CCR4 is measured by serially diluting eachpurified antibody prior to staining Cf2-CCR4⁺ cells. The ability of thepurified antibodies to bind to other CCR4 expressing cell lines, whichexpress different conformations of CCR4, is also examined. Cell linesused in this procedure include Mac-1 cells, 293T-CCR4⁺ cells,CEM.NKR.CCR4⁺ cells, HeLa-CD4⁺/CCR4⁺ cells and Ghost-CD4⁺/CCR4⁺ cells.

Example 22: Affinity Measurements of Anti-CCR4 scFvFcs and IgGs UsingBiaCore

The equilibrium dissociation constants (K_(D)) of 1567 scFvFc and IgGvariants are determined by surface plasmon resonance on a BIACORE T-100instrument using the BIACORE Pioneer L1 chip, which has been designed towork with lipid bilayers for the study of trans-membrane proteins. Thisbiosensor chip has a carboxymethylated dextran matrix that has beenmodified with lipophilic substances and designed to capture liposomesrapidly and reproducibly (Stenlund P. et al. Anal Biochem 2003,316:243-250; Navratilova I. et al. Anal Biochem 2006, 355:132-139;Navratilova I. et al. Anal Biochem 2006, 353:278-283; Navratilova I. etal. Anal Biochem 2005, 339:271-281). The capturing process permits theliposome to retain the lipid bilayer structure in such a way thatinjected liposomes can carry membrane-anchored molecules that protrudeon one, or both sides of the lipid bilayer. The optimal conditions forimmobilizing the Pioneer LI chips with purified hCCR4 PMPLs ispre-determined by using three different coupling buffers at standardrunning conditions. Association rates are measured using a constant flowof 5 μl/min and sFvFc and IgG concentrations ranging from 5×10⁻⁶ to1×10⁻⁹M. K_(on) is determined from a plot of ln (dR/dt)/t vs.concentration (Karlsson R. et al. J Immunol Methods 1991, 145:229-240).Dissociation rates are measured using a constant flow of 25 ul/min andscFvFc/IgG concentration of 1.0×10⁻⁶M. K_(off) is determined during thefirst 30 seconds of dissociation. K_(D) is calculated as K_(off)/K_(on).

Example 23: In Silico Modeling of 1567 VH and VL

The humanization of the mouse Mab 1567 is approached from severaldifferent directions to ensure the optimal result, which is an antibodywith a resulting binding affinity of ≤1 nM⁻¹. In silico modeling ofVH1567 is modified, with particular attention paid to the amino acids atthe framework-CDR boundaries, which can be altered to provide greaterflexibility in the binding site. New mutants of the initially modeled VHare produced by site-directed mutagenesis and retested for their bindingaffinity.

In silico modeling of the VL is completed in a similar manner to VH, andthen used as a template for the humanization processes described inherein, including VH swapping studies. AMBER software (Assisted ModelBuilding using Energy Refinement) is used for this modeling work.

Example 24: Epitope Mapping of Mab 1567 Using hCCR4 Mutant or ChimericProteins

For all subsequent epitope mapping descriptions, the humanized 1567antibody is evaluated in the form of highly-purified soluble scFvFc andIgGs isolated from supernatants of transfected 293T cells. The methodsused for identifying the epitope region are described here.

A series of hCCR4/hCCR2 chimeras by blunt-end ligation are constructed.hCCR2 has 45.6% a.a. sequence homology with hCCR4. A similar approachhas been established for the construction of hCCR5/hCCR2b andhCXCR4/CXCR2 chimeras (Lee B. et al. J Biol Chem 1999, 274:9617-9626;Baribaud F. et al. J Virol 2001, 75:8957-8967). The N-terminus (Nt) andextracellular loop (ECL) regions 1, 2 and 3 of hCCR4 and hCCR2 are PCRamplified and blunt ligated. Using the designation of “4444” torepresent the wild type (WT) CCR4 Nt, ECL1, ECL2 and ECL3, and “2222” torepresent the WT CCR2 Nt, ECL1, ECL2 and ECL3, a complete series ofchimeras are constructed that contain different Nt or ECL segments ofthe two chemokine receptors (e.g. 2444, 4244, 4424, etc.). This strategyenables the identification of linear, conformationally sensitive, anddiscontinuous epitopes on CCR4. All WT and chimeric expression plasmidscontain a 3 amino acid tag to normalize surface protein expression byFACS analysis. For these studies, 293T cells are transfected with WT orchimeric constructs. Purified 1567 antibody is contacted to cells at aconcentration of 50 ug/ml, followed by the addition of a fluorescentlylabeled antibody, phycoerythrin (PE)-labeled anti-IgG. The mean channelfluorescence (MCF) is used to compare the levels of antibody binding.Results are normalized for the MCF obtained for antibody binding to WTCCR4 (normalized as 100%) after subtraction of the background MCFobtained against empty vector-, pcDNA3,-transfected cells (normalized as0%).

A series of N-terminal deletion mutants (Δ4, Δ8, Δ12 and Δ16) are usedto define the precise region to which Mab 1567 binds. Furthermore aseries of CCR4 point mutations, located within the Mab 1567 bindingregion, are generated by substituting the uncharged residue, alanine,for charged amino acids to identify residues that are important forbinding. Charged amino acids that serve a critical binding function inthe WT CCR4 epitope region demonstrate a loss-of-function, or loss ofbinding, when substituted for alanine. In addition to FACS analyses,Western blotting techniques are used to determine if the Mab 1567antibody, when bound to WT, chimeric, or mutant hCCR4, recognizes linearor conformational epitopes (Lee B. et. al. J Biol Chem 1999,274:9617-9626).

Epitope mapping studies are performed by analyzing the binding ofMab1567 to mouse and guinea pig CCR4, which show high homology to humanCCR4, except in the N-terminal region (FIG. 15). CCR4 is also cloned andPCR amplified from PBMCs sampled from cynomolgus and rhesus macaqueshoused within the New England Regional Primate Research Center. PCRprimers are designed against the 5′ and 3′ flanking regions of humanCCR4 in order to obtain unbiased sequence data at the beginning and endof the open reading frame (ORF). In all cases, the ORFs are stablyexpressed in Cf2 cells following G418 selection. The cross-reactivity ofMab 1567 to other human CCR and CXCR chemokine receptors will be tested.Exemplary chemokine receptors used to test cross-reactivity include:CXCR1, CXCR2, CXCR4, GPR1, GPR15, Str133, Dez, Apj CX3CR1, CCR1, CCR2B,CCR3.

Example 25: Mab 1567-Mediated Inhibition of CCR4 Ligand Binding

Recombinant CCL17 and CCL22 binding to WT and chimeric CCR4 arecharacterized to identify critical ligand-binding domains. First,CCL17-Fc and CCL21-Fc fusion proteins are constructed. FACS analyses areperformed in order to detect receptor-ligand binding events using aFITC-conjugated anti-human IgG1. Transfected cells are incubated withthe chemokine fusion protein, ligand, that binds to these cells viaeither the WT or chimeric CCR4 receptor. Stable Cf2-CCR4⁺ cells are usedto test the ability of the Mab1567 antibody to block binding of theligands, CCL17 and CCL22, to CCR4-expressing cells. The ability ofMab1567 to block binding is determined by either the loss of medianfluorescence intensity (MFI) or the percentage of fluorescently-labeled,or positive, cells. Positive controls include unlabelled recombinantCCL17 and CCL22 (Bioclon, Inc.). Non-specific binding is determined asMFI bound in the presence of a 100-fold excess of unlabeled ligand.

Example 26: Mab1567 Inhibition of CCL17/CCL22-Mediated PBMC Chemotaxisand Intracellular Calcium Mobilization

Chemotaxis assays are performed with PBMCs in 24-well plates withtranswell inserts of 5 μm pore size (Costar, Cambridge, Mass.). 5×10⁵cells are plated on the cover membrane in RPMI-1640 medium containing0.4% fetal bovine serum and incubated at 37° C. For these studies afixed optimal concentration of chemokine is used and serial dilutions ofthe Mab1567 are tested. Similar procedures are performed to evaluate theability of the Mab1567 to block chemokine-mediated intracellular calciummobilization. Calcium mobilization assays in PBMCs are performed usingFURA-2 (Molecular Probes) (Juremalm M. et. al. Clin Exp Allergy 2005,35:708-712). Fluorescence measurements are taken with a Hitachi F-2000fluorescence spectrophotometer using the dual-wavelength optionalfunction at wavelengths 340/380 nm (excitation) and 505 nm (emission).Data points are collected every 500 ms and recorded as the relativeratio of fluorescence excited at 340 and 380 nm. The ability of theMab1567 to block CCL17/CCL22-mediated chemotaxis and intracellularcalcium mobilization is also tested in Sezary cells from patients. Theresults show that the Mab1567 antibody blocks chemotaxis mediated byCCL22 (FIG. 8).

Example 27: Mab1567-Mediated Depletion of CCR4-Bearing Cells

The ability of Mab1567 to deplete CCR4-bearing cells is tested inmultiple in vitro and in vivo assays. For the in vitro assays, Mab1567is secreted from transiently-transfected 293T cells and purified byprotein A chromatography. For in vivo assays, the experimental mice aremanipulated to produce Mab1567 antibody via AAV-8-mediated genetransfer. A method was devised to assess the in vivo depletion of PBMCby utilizing magnetic beads to efficiently eliminate CCR4⁺ cells fromPBMC in vitro. This strategy yields a meaningful result without priorknowledge of which potential Mab 1567-mediated mechanism executes CCR4depletion in vivo.

In this example, Dynabeads® Pan Mouse IgG magnetic beads (Dynal product#110.41) are produced with a coating of a single Mab that recognizes allclasses of mouse IgG, but does not cross-react with any class ofhuman-derived antibodies. These beads are “charged” with amouse-anti-human IgG1 Mab (SouthernBiotech product #9052-01), so thatthey bind to any cell that has been coated with human IgG1, or in thiscase, any cell that binds the Mab 1567.

The granulocyte types, naïve T cells, B cells or NK cells are used asnegative controls in this study because it is known that these cellpopulations do not express CCR4 (Campbell J. J. et al. Nature 1999,400:776-780). Based upon levels of CCR4 expression, Mab1567 is expectedto deplete a significant proportion of memory T cells and Treg cells.Recent publications by those skilled in the art suggest that this typeof cell is responsive to CCR4 ligands (Hirahara K. et al. J Immunol2006, 177:4488-4494; Iellem A et al. J Exp Med 2001, 194:847-853) (FIGS.11 and 12). Treg cells are believed to play a role in immunotolerance toneoplastic cells (Abbas A, Lichtman, A H. Cellular And MolecularImmunology, Updated Edition (ed 5). Elsevier Saunders 2005). Thereforeelimination of these cells mediated by Mab 1567 supplements existingmechanisms that directly eliminate leukemic CTCL cells and dramaticallyincrease the effectiveness of Mab1567 in arresting the progression ofCTCL.

It is not unprecedented that chemokine receptors exist withpost-translational modifications in different cell types (Hill C. M. etal. Virology 1998, 248:357-371). The results of these depletion assaysare screened to find these differences, as well as conformationalheterogeneity within CCR4 proteins.

Example 28: Mab1567-Mediated Apoptosis Induction of CCR4⁺ Cells In Vitro

The ability of MAb1567 to induce apoptosis of CCR4-expressing cellsafter cross-linking with a secondary antibody is examined. Mac-1 cellsin exponential growth phase (grown in supplemented RPMI with 10% bovineserum) are incubated with Mab1567 or with the negative control IgG1 hMabon ice. These cells are then washed and incubated again on ice for 1hour with F(ab′)2 fragments of goat-anti-human IgG (heavy and lightchain specific, Jackson ImmunoResearch product #109-006-003). Next,cells are centrifuged, washed, and resuspended in Mac-1 culture mediumat 37° C. Incubation proceeds for 2 hours to allow any signaltransduction to occur that might result from CCR4 cross-linking. Afterincubation, the cells are washed and resuspended in cold medium.

Whether or not cross-linking of CCR4 induces apoptosis is assessed usingan annexin V-based apoptosis detection procedure (Annexin V-FITCApoptosis Detection Kit II, BD Pharmingen product #556570). This assayis based on the finding that annexin V binds to phosphatidylserine (PS),which is only associated with the internal membrane components ofhealthy cells, but, as a result of compromised membrane homeostasis,appears on the cell surface during apoptosis. Thus, a cell that stainswith FITC-conjugated annexin V but is still intact (as assessed bypropidium iodide (PI) exclusion) and is in the early stages ofapoptosis. Cells in late stages of apoptosis stain with bothFITC-conjugated annexin V and PI.

Other CCR4-expressing cell-types available for these assays (e.g. stableCf2-CCR4 and 293T-CCR4 cells) are tested in parallel with Mac-1 cells tocircumvent complications arising from a potential insusceptibility ofMac-1 cells to apoptosis. Furthermore, primary PBMCs are included inthis assay (although a negative control other than CCR5, e.g. humananti-HA 3B3 IgG, is utilized in this case), and, PBMC from leukemic CTCLpatients are tested.

Example 29: Mab1567-Mediated Depletion of CCR4⁺ Cells by ADCC In Vitro

The ability of Mab1567 to target CCR4-expressing cells for destructionvia ADCC is assessed. The DELFIA EuTDA cytotoxicity kit, as described inFIGS. 9 and 10, is used to detect cytotoxicity. Of particularphysiological relevance, the ability of Mab1567 to trigger ADCCresponses with human effector cells is examined. It is necessary tooptimize conditions separately for human PBMC and mouse splenocytes. Theanti-CD30 antibody, already known to robustly bind Mac-1 cells(Kleinhans M. et al. Blood 2003, 101:1487-1493), is employed foroptimization of target: effector ratios and other parameters requiredfor utilization of the DELFIA kit.

Mac-1 cells are treated with Mab1567 or a negative control Mab. Thesecells are washed and resuspended in culture medium with the optimizednumber of human PBMCs and incubated for 4 hrs at 37° C. All conditionsare repeated in triplicate wells. The level of cell death achieved foranti-CCR4-treated cells is compared to that of control-treated cells.The control for spontaneous release is prepared by culture of thelabeled cells only and the control for maximum release is obtained byadding lysis buffer to the labeled cells.

Other cell types including stable Cf2-CCR4 and 293T-CCR4 cells areexamined to determine the effect of CCR4 surface density on cell killingbecause these cells express lower levels of CCR4 compared to Mac-1cells. Primary PBMCs are tested in this assay (anti-HA 3B3 Mab isutilized at negative control in this case), and, PBMCs from leukemicCTCL patients are tested as target cells when available.

Example 30: Mab1567-Mediated Depletion of CCR4⁺ Cells byComplement-Mediated Lysis In Vitro

The ability of Mab1567 to target CCR4-expressing cells for destructionby the complement system is examined. Complement-mediated cell damage isassessed by the DELFIA EuTDA cytotoxicity kit. Guinea pig complement(lyophilized, Rockland Immunochemicals product # C200-0005) is preparedfresh for each experiment. Mac-1 cells are coated with either Mab1567 ornegative control Mab, washed, and resuspended in low-serum medium in96-well plates. Freshly prepared guinea pig complement is added to eachwell at the optimal concentration, and the plates are incubated at 37°C. for 30 minutes. The level of cell death for anti-CCR4-treated andcontrol-treated cells is compared.

Example 31: Mab1567-Mediated Depletion of CCR4⁺ Cells by ADCP In Vitro

The ability of Mab 1567 to enhance the phagocytic activity of monocytesand macrophages, a process called antibody-dependent cellularphagocytosis (ADCP), is assessed. Human monocytes/macrophages areseparated from human PBMCs by negative selection with anti-CD3-, CD19-,and CD56-based magnetic cell sorting. Mac-1 target cells are incubatedwith the lipophilic fluorochrome PKH26 (Sigma), mixed in a 1:5 ratiowith monocytes/macrophages, and co-cultured with either Mab1567 orcontrol Mab at 10 ug/ml. Following a 1 hour incubation at 37° C., thecells are harvested by EDTA, residual Fc receptors are blocked with ratserum (DAKO), and the monocyte/macrophages are stained withFITC-conjugated anti-CD14 Mab. The spontaneous phagocytosis iscalculated from cultures lacking Mab1567 but containing control Mab. Themixture of PKH26-labeled Mac-1 cells and human monocyte/macrophages isstained also with FITC-conjugated anti-CD14 Mab, without pre-incubation,and used as a control. Double-positive cells (PKH26/FITC) representMac-1 cells phagocytosed by the monocytes/macrophages.

Example 32: Mab1567-Mediated Depletion of CCR4⁺ Cells by BlockingLigand-Mediated Signaling Through CCR4 In Vitro

Primary CTCL cells are difficult to grow in culture. In fact, reliabletissue-adapted CTCL lines are not currently available. Although,malignant cells can now be directly isolated from skin lesions. Thisculture obstacle is likely due to the fact that growth factors availablein vivo have not been adequately provided by the culture conditionstested to date. CCR4 is remarkably persistent on leukemic CTCL cells,despite frequent loss of other crucial skin-homing molecules. Oneintriguing explanation for this persistence might be a dependence onCCR4 signaling to provide necessary pro-growth signals for in vivo.Therefore, the ability of CTCL cells to survive in culture was tested inthe presence of soluble CCR4 ligands. If ligand-CCR4 interactionspromote in vitro survival, the ability of Mab1567 to block CCL17 andCCL22 binding to CCR4 can be used to inhibit growth of leukemic CTCL invivo. The experiments used to test this therapeutic avenue are describedbelow.

Primary CTCL cells isolated from leukemic CTCL patients are cultured insupplemented RPMI with 10% bovine serum among individual wells in24-well plates with either recombinant CCL17 or CCL22 present at variousconcentrations, or in the absence of chemokines. Each chemokine istested at concentrations 0.1×, 1× and 10× their interaction K_(D) withCCR4. The cultures are monitored visually for differences among wellswith regard to cell number. If greater numbers of cells are detected inwells that contain one of the CCR4 ligand concentrations, widerchemokine concentration ranges are tested in order to optimize the invitro culture conditions for primary leukemic CTCL cells. Once theoptimal CCR4-ligand-dependent growth conditions are established forprimary leukemic CTCL cells, Mab1567 are tested at variousconcentrations in individual culture wells to determine if Mab treatmentcan block this proliferative effect. Similar experiments are performedwith skin-derived CTCL cells.

Example 33: Mab1567-Mediated Depletion of CCR4⁺ Cells In Vivo

The ability of Mab1567 to prevent growth and/or mediate destruction ofCCR4-expressing cells is assessed using a xenograft SCID/Beige mousemodel. Based on the in vitro data generated above, the most potenthumanized Mab1567 is tested in the mouse model of CTCL. For thesestudies, both antibody gene transfer and protein injection methods areused to deliver therapeutic levels of Mab1567 in order to assess itsability to prevent tumor growth in a pre-implantation and post-tumorimplantation Mab treatment study.

Example 34: AAV-8-Mediated Gene Transfer

A remarkably powerful technique has recently been reported, and modifiedby the Marasco laboratory, that can allow human Mabs to be expressed attherapeutic levels for prolonged periods of time (several months)following systemic delivery by a single intravenous injection of genetransfer vector (Marasco W A. Nat Biotechnol 2005, 23:551-552)(see FIG.16). Specifically, gene transfer is accomplished using a newly reportedrAAV serotype 8 (AAV-8) vector. Recently isolated from rhesus macaques,this vector has the capacity to transduce hepatocytes and skeletalmuscle with very high efficiency when delivered by portal vein injectionor intravenous infusion (Nakai H et al. J Virol 2005, 79:214-224; Gao G.P. et al. Proc Natl Acad Sci USA 2002, 99:11854-11859). A recent reportby Fang et al. (Nat Biotechnol 2005, 23:584-590) demonstrated thatremarkably high levels (>1,000 ug/ml) and long-term expression (>140days) of an anti-VEGR2 Mab could be achieved in mice and demonstratedtherapeutic efficacy against two tumor cell lines in two mouse tumormodels.

Example 35: AAV-8-Mediated Gene Transfer In Vitro

The AAV vector (pTR-UF20, gift of Dr. N. Muzyczka, Univ of Florida) hasbeen modified to accommodate two types of antibodies, the bivalentscFv-Fc fusion proteins and whole human IgG1 Mabs. The latter utilizesthe furin-2A-self cleavage cassette which results in stoichiometricallyequivalent amounts of heavy and light chains. In both antibodyconfigurations, interchain disulfide bonding is intact and fullyfunctional bivalent molecules are secreted. This plasmid uses the CMV IEenhance, chicken β-actin promoter and intron sequences to drive highlevels of antibody expression.

For the studies outlined here, rAAV vectors encoding Mab1567 IgG1 areused. The AAV-Mab1567 vector plasmid is co-transfected with theAAV2rep/AAV8cap packaging plasmid (p5E18-VD2/8, gift from Dr. J. Wilson,Univ of Penn)(Gao G. P. et al. Proc Natl Acad Sci USA 2002,99:11854-11859) and the mini-adenovirus helper plasmid (pXX6-80, giftfrom Dr. J. Samulski, Univ North Carolina)(Xiao X. et al. J Virol 1998,72:2224-2232) into subconfluent 293 cells using a calcium phosphatemethod. 48 hours after transfection, cells are harvested using PBS/EDTA(10 mM) and lysed by three freeze/thaw cycles in cell lysis buffer.Lysates are treated with 250 U/ml benzonase for 15 min at 37° C. andcellular debris is removed by centrifugation. The cleared cell lysate isfractionated by ammonium sulfate precipitation and the rAAV virions areisolated on two sequential CsCl gradients. The gradient fractionscontaining rAAV are dialyzed against sterile PBS containing CaCl2 andMgCl2, and stored at −80° C. Viral titers are determined by dot-blotanalysis (Fang J et al. Nat Biotechnol 2005, 23:584-590). Each rAAVvirion preparation is first tested in vitro on transduced 293 cells. Thetime course and level of antibody secretion are then analyzed by humanIgG capture ELISA with reagents that recognize the Fc of IgG1, by cellbased ELISA titers on stable CCR4⁺ cells, and by Coomassie blue stainingon non-reducing and reducing SDS-PAGE gels of antibody that is purifiedfrom the supernatants by protein A-agarose beads.

Example 36: Prevention of New Tumor Implantation in a CTCL Mouse Model

The timing and levels of Mab1567 antibody expression in vivo areanalyzed in pilot studies in SCID/beige mice. Briefly, groups of 8 mice,comprising a test cohort, are injected by the tail vein withrAAV8-Mab1567 (1×10¹¹, 2×10¹¹ or 4×10¹¹ vector genomes (vg)/mouse) andantibody levels are analyzed over time by human IgG capture ELISA. Miceare subsequently bled by alternate retro-orbital puncture at eachscheduled time point for up to 4 months for analysis of recombinantantibody expression. The results of these studies determine the optimalvector dose of Mab1567 that is required to achieve an expectedtherapeutic dose and the duration after vector administration, for whichthat dose should be achieved.

For the mouse tumor models, female SCID/beige mice (n=10-12 in eachgroup) are injected with rAAV8-Mab1567 or rAAV8-control Mab virions viatail vein in 200 ul PBS at the optimal dose, as determined above. Micein the negative control group are treated with the same dose of ananti-CCR5 Mab-encoding rAAV8 vector. To monitor human IgG1 levels, miceare bled weekly by alternate retro-orbital puncture. When steady statehas been achieved, circa 21 days, the animals are inoculated withsingle-cell suspensions of the luciferase-expressing human cutaneousCCR4⁺ ALCL line Mac-1 (2×10⁷ cells) into the left flank of the mouseusing a 13-gauge trocar. These cell lines have already been produced bytransduction with MuLV-luciferase virus and stable high luciferaseexpressing subclones have been established. The size of the subcutaneoustumors are measured with a caliper and tumor volumes are recordedaccording to the formula V=d×D×π/2, where d is the smaller diameter andD the larger diameter. Mice are monitored for tumor development andprogression by both caliber measurement and Xenogen imaging. The latter,which allows monitoring of metastatic disease in living animals, isperformed twice each week following intravenous injection of 50 mg/mlD-luciferin. Treated and control mice are eventually euthanized andnecropsied for evidence of tumors. Post-mortem analysis of tumorformations includes histology and immunohistochemistry. Mice areeuthanized when tumor diameter reaches 1.5 cm or when moribund. Thisprocedure was repeated with additional cohorts of mice comprising atotal of 24 mouse subjects.

In order to determine if Mab1567 has potent anti-tumor activity in vivo,non-parametric and parametric methods of analysis are used to comparehuman IgG1 levels between the negative control rAAV8-IgG1 and theexperimental rAAV8-Mab1567 group. An adjustment is made for multiplepair-wise comparisons. It is anticipated that this experimental approachestablishes that Mab1567 has potent anti-tumor activity in vivo. Ifpotent anti-tumor activity is not observed and this result is notattributable to low serum levels of the Mab1567 protein, theconventional method of delivering antibody by intraperitoneal injectionis preferred since this allows peak and trough antibody levels to occurwhich may have a positive effect on the ability of the immune system toclear the antibody bound tumor cells through FcγR-mediated mechanisms.In this event, the purified Mab1567 protein is injected byintraperitoneal injection 20 mg/kg scFvFc twice each week for up to 4weeks. Alternatively, the heavy chain isotype of Mab1567 is changed tohIgG3 in order to increase complement-mediated tumor clearance. In thisevent, the isotype switch is performed and AAV8-Mab1567 virions are usedto deliver the hIgG3 homolog.

Example 37: Destruction of an Existing Tumor in a CTCL Mouse Model

Destruction of an existing tumor in an established SCID mouse CTCLxenograft model is described. It is uncertain at the present timewhether or not the kinetics of in vivo antibody production in the AAV8gene transfer system are sufficiently robust to achieve therapeuticantibody concentrations in the time frame required to inhibit growth ofexisting tumors. Therefore, AAV8 gene transfer, protein injection, and acombination of these methods are used to deliver Mab1567 to mice withestablished tumors and the results of each attempt are compared.

These studies are conducted as described in Example 32 (SCID/beige micein groups of 10-12) except that gene delivery and protein injectionbegin on days +1 and +5 after tumor implantation in order to assess theability of Mab 1567 to inhibit early versus late tumor growth.Specifically, on day +1 (early tumor) or day +5 (late tumor), theanimals are injected intravenously (i.v.) with AAV8-Mab1567 virions(single optimal dose) or intraperitoneally (i.p) with Mab1567 proteintwice each week for the first 4 weeks. A third treatment group alsoreceives both treatments beginning on day +1 or +5 however, in thisgroup, only two i.p. injections of Mab1567 protein are used whichprovides an initial high serum concentration of antibodies until thegene transfer antibody reaches a therapeutic level. Control mice receiveidentical treatments with an isotype control Mab. Tumor growth ismonitored in live animals by caliper measurements and Xenogen imaging.Histology and immunohistochemistry are performed on tissue harvestedfrom sacrificed animals as described in the “detailed description”section of this application. This procedure was repeated with additionalcohorts of subjects comprising 72 in total.

In order to determine if Mab1567 is able to inhibit the growth of earlyand late established CCR4⁺ ALCL tumors, non-parametric and parametricmethods of analysis are used to compare the three groups treated withAAV8-Mab1567 vector, Mab1567 protein, or both AAV8-Mab1567 and Mab1567protein, respectively. An adjustment is made for multiple pair-wisecomparisons. It is anticipated that the experimental results obtainedfrom these procedures demonstrate that Mab1567 is able to inhibit thegrowth of early and late established CCR4⁺ ALCL tumors. However, theCCR4⁺ ALCL cell line may not be predictive of the in vivo growthbehavior of CCR4⁺ CTCL lines and the inhibition of their growth byMab1567.

Example 38: Mab1567 Production in Chinese Hamster Overy (CHO) Cells

A high secretor CHO (DG44) transfectoma cell line is established andtens of milligrams quantities of Mab1567 are purified. The humanimmunoglobuin IgG1 kappa (TCAE5) expression vector is used to targetmammalian loci that support high levels of expression. This vectorencodes immunoglobulin heavy and light chain genes, the dihydrofolatereductase (DHFR) gene, and the dominant selectable marker neomycinphosphotransferase (Neo) gene. As a result of intentional impairment ofthe Kozak sequence surrounding the Neomycin initiation codon, includedto create a fully impaired Kozak sequence, most single copy integrantsdo not express enough Neo to survive selection (Kozak M. J Mol Biol1987, 196:947-950). The result is that the overall number of G418resistant cells is greatly reduced, thereby facilitating screening. Ahigher percentage of the clones surviving selection are those in whichthe impaired Neo gene has been integrated into “hot spots” with thegenome, which concomitantly yield very high levels of linked geneexpression (Barnett R. L. et al. Antibody Expression and Engineering.Edited by Wang H Y IT, 1995, pp 28-40). Once isolated, transfectantswhich display a very high level of immunoglobulin protein production areinduced to undergo gene amplification by selection with increasingconcentrations of methotrexate (MTX) (5 nM→50 nM→500 nM) for thedihydrofolate reductase gene (Kaufman R J, Sharp P A. J Mol Biol 1982,159:601-621). As the DHFR gene copy number increases throughamplification, there is a parallel increase in the closely linkedimmunoglobulin gene copy number with an accompanying rise inimmunoglobulin production. Amplification of initially very high levelexpression clones yields cells producing even greater levels ofimmunoglobulin protein from a minimal number of gene copies (Barnett R.L. et al. Antibody Expression and Engineering. Edited by Wang H Y IT,1995, pp 28-40). At the 500 nM stage, the selected amplificants arereadapted to grow in spinner flasks. During this time, transfectomaantibody is purified from the supernatants over protein A. When the cellis producing 100 pg/cell/day and has a doubling time of 36 hours orless, it is considered a production cell line and a Parent Seed Stock isprepared.

Example 39: Mab Production in YB2/0 Cells

One IgG molecule contains two N-linked oligosaccharides sites in its Fcregion (Rademacher T. H. et al. Presented at the Biochem Soc Symp,1986). The general structure of N-linked oligosaccharide of IgG iscomplex-type, characterized by a mannosyl-chitobiose core(Man3GlcNAc2-Asn) with or without bisecting GlcNAc/L-fucose (Fuc) andother chain variants including the presence or absence of Gal and sialicacid. In addition, oligosaccharides may contain zero (G0), one (G1), ortwo (G2) Gal. ADCC requires the presence of oligosaccharides covalentlyattached at the conserved Asn²⁹⁷ in the Fc region and is sensitive tochange in the oligosaccharide structure. Recent studies have shown thatengineering the oligosaccharides of IgGs may yield optimized ADCC. Inparticular, the absence of fucose, but not the presence of galactose orbisecting N-acetylglucosamide of human IgG1 complex-typeoligosaccharides, has been shown to play the critical role of enhancingantibody-dependent cellular toxicity (Shinkawa T. et al. J Biol Chem2003, 278:3466-3473). Human IgG1 produced by rat hybridoma YB2/0 cellsshowed extremely high ADCC at more than a 50-fold lower concentration ofthose proteins produced by CHO cells. YB2/0 cells expressed a lowerlevel of FUT8 (α1,6-fucosyltransferse gene) and produced IgG1 of lowerFuc content compared to CHO cells (Shinkawa T. et al. J Biol Chem 2003,278:3466-3473).

The hypothesis that ADCC contributes to Mab1567-mediated immunodepletionof CCR4⁺ ALCL tumors in SCID mice is evaluated by comparing the in vitroand in vivo killing effects of anti-CCR4 Mab antibody produced in CHOand YB2/0 cells. An efficient transfection procedure for YB2/0 cells(Amixa kit) has been established. Thus, a high secretor cell line fromYB2/0 is developed, as described for the CHO cell lines above. Again,when the cell is producing 100 pg/cell/day and has a doubling time of 36hours or less, it is considered a production cell line and a Parent SeedStock is prepared. Transfectoma antibody is purified from thesupernatants over protein A. Tens of milligrams of total Mab from thehigh-secretor CHO and YB2/0 cell lines are produced for further in vitroand in vivo target validation studies.

Example 40: ADCC of ATCL/CTCL Cells In Vitro by Mab1567 withDifferential Fucose Content

Mab1567 produced in Examples 35 and 36 is used to evaluate the role ofADCC in tumor cell killing in vitro. In brief, The cytotoxic activity ofhuman PBMCs is determined using the DELFIA EuTDA cytotoxicity kit. Fiftymicroliters of target cells at 2×10⁵ cells/ml are added to 96-well“U”-bottomed microtiter plates (Nunc) to achieve effector/target (E: T)ratios varying from 25:1 to 0.7:1. PBMCs are isolated with Ficoll-PaguePlus from buffy coats. Effector cell number in all assays is calculatedbased on the total number of mononuclear cells. Varying doses (50, 10,1, 0.1, 0.01 μg/ml final concentration) of Mab1567 or negative controlisotype matched Mab are tested. Background killing activity of targetcells is concurrently measured by incubation of PBMCs with target cellsin the absence of Mab. Each sample is measured in triplicate. Controlsinclude spontaneous release (medium alone) and total release (10%Tween-20/PBS). It is expected that the YB2/0-produced Mab is more activethan the CHO-produced Mab in this ADCC assay.

Example 41: ADCC in Tumor Prevention and Destruction in a CTCL MouseModel

Potent anti-tumor activity of Mab1567 in the SCID/Beige model with Mac-1xenografts were shown. Remarkably, a profound effect was seen despitethe fact that these mice are devoid of NK cells which mediate ADCC. Theability of the Mab 1567 to prevent and destroy tumors is established inan alternative mouse model by using severe combined immunodeficiency(SCID) mice in order to evaluate the in vivo contribution of ADCC intumor destruction. To evaluate the role of ADCC in prevention of newtumor implantation in vivo, female SCID mice (n=10-12 in each group)receive an intraperitoneal injection of either 20 mg/kg, 10 mg/kg, 5mg/kg or 1 mg/kg IgG beginning 24 hours before tumor implantation andthen twice each week for 4 weeks. Because it is expected that bothantibodies are able to cause immunodepletion of the tumor cells, it isexpected that this dose response comparison of the CHO and YB2/0produced IgG1 Mabs is able to detect differences in the in vivo potencyof the two Mabs. An isotype matched human IgG1 control Mab produced fromthe same cells will serve as controls (n=5-6 in each group). Theseprocedures were repeated with additional cohorts of subjects comprisinga total of 100-120 mice.

The ability of Mab1567 to destroy existing tumors in vivo is evaluatedwith the optimal dose of Mab1567 determined in the pre-implantationtreatment model. In brief, one fixed and equal concentration of each Mabis used. The ability of the Mab to inhibit early and late tumor growthis evaluated. Specifically, on day +1 (early tumor) or day +5 (latetumor) after tumor implantation, female SCID mice (n=10-12 in eachgroup) receive an intraperitoneal injection of a fixed and equal dose ofCHO or YB2/0 Mab twice each week for 4 weeks. An isotype matched humanIgG1 control Mab produced from the same cells serves as the control(n=5-6 in each group). It is anticipated that the results of this studyare parallel to the results described above. In both models, monitoringof tumor growth by caliper measurements, Xenogen imaging in live animalsand by histology and immunohistochemistry in sacrificed animals isperformed as described in Examples 33 and 34. These procedures wererepeated with additional cohorts of mice comprising a total of 60subjects.

Following the conclusion of the above studies, the most potent antibodyis chosen and the stable high secretor cell line is transferred to theNational Cell Culture Core (NCCC). The NCCC is contracted to establish anon-GMP cell bank from the Parental Seed Stock of high-secretor CHO orYB2/0 cell line and to produce 20 grams cGLP grade Mab1567 for animalstudies. Under contract, the cells are thawed, screened for mycoplasma(GLP), a 10 vial cell bank (GLP) is made, and production optimizationwork (media screening using the micro-bioreactor) is performed.Supernatant produced from the hollow fiber bioreactor is furtherprocessed using affinity chromatography to produce bulk purifiedantibody with low levels of endotoxin.

Example 42: Mab 1567-Mediated Immunodepletion of CCR4⁺ T Cells from thePeripheral Blood and Lymphoid Tissues of Non-Human Primates

The cynomolgus monkey (Macaca fascicularis) is widely used as an animalmodel for analyzing immunity, hematopoiesis, infectious diseases,transplantation, and toxicology, because this macaque species isphylogenetically proximate to humans (Vugmeyster Y et al. Cytometry A2003, 52:101-109; Vugmeyster Y. et al. Int Immunopharmacol 2003,3:1477-1481). In particular, cynomolgus monkeys have been extensivelyused to study therapeutic agents aimed at B-cell depletion, such asanti-CD20 (Rituximab) (Reff M. E. et al. Blood 1994, 83:435-445) andanti-CD40 Mabs (Boon L. et al. Toxicology 2002, 174:53-65) and inpreventing allograft rejection with anti-CTLA-4 (Palmisano G. L. et al.Clin Exp Immunol 2004, 135:259-266). Rhesus macaques (Macaca mulatta)have been infused with anti-CD80 (B7-1), anti-CD86 (B7-2) Mabs to assessallograft rejection as well (Vugmeyster Y et al. Cytometry A 2003,52:101-109). To obtain additional data to support the hypothesis thatMab1567 can be used for the immunodepletion of CD4⁺/CCR4⁺ CTCLs,preliminary feasibility studies to assess the in vivo immunodepletion ofCD4⁺/CCR4⁺ T-cells from peripheral blood (pb), bronchoalveolar lavage,bone marrow (BM), and lymphatic tissue are performed following systemicadministration of Mab1567 in either cynomologus or rhesus macaques.

All studies are performed at the New England Primate Research Center.Macaques weighing 4-6 kg are used in the studies with one animalreceiving each dose in the dose escalation studies. The most potent formof Mab1567, either CHO- or YB2/0-produced Mab, is used. Preliminary invitro studies are performed to confirm that Mab1567 binds to CD4⁺/CCR4⁺T cells by FACS staining. Subsequently, each animal receives a singledose infusion of Mab given intravenously corresponding to 0.05 mg/kg,0.5 mg/kg, 1 mg/kg, 5 mg/kg and 10 mg/kg. An isotype-matched control Mabis given to four control animals at 10 mg/kg. Pre-treatment peripheralblood is obtained. Peripheral blood samples are also collected on days1, 2, 4, 8, 15, 29, and subsequently thereafter at biweekly intervalsuntil completion of the study on day 90. Lymph node biopsies from theinguinal nodes are taken at days 15 and 29 with cell preparationsstained for quantification of lymphocyte populations by flow cytometry.Percent depletion of CD4⁺/CCR4⁺ in anti-CCR4 Mab treated animals iscalculated by the formula: % depletion=100−% CD4⁺ CCR4⁺ cells in treatedlymph nodes divided by % CD4⁺/CCR4⁺ cells in control Mab treatedanimals. Other Mabs are used to assess the effect of anti-CCR4 Mabtreatment on B cells (anti-CD20), T cell subsets (anti-CD3, CD4, CD8,CD45RA, CD45RO) including Tregs (CD25, FoxP3), macrophages (CD68,CD163), and others.

The immunophenotypic composition of bronchalveolar lavage cells isperformed to evaluate the effect of Mab1567 on pulmonary immune cells.Briefly, animals are anesthetized with ketamine HCl and 0.4 mm diameterbronchoscope is used to infuse a 20 ml aliquot of sterile physiologicsaline (PBS) (Hendricks E. E. et al. J Infect Dis 2004, 189:1714-1720).The fluid is removed and the procedure is repeated 3-4 times. The cellsare pelleted and washed 3 times with PBS before staining with monoclonalantibodies and FACS analysis. Pre-treatment values are compared tosamples taken on days 2, 8, and 29.

In order to evaluate alterations in the T cell composition of lymphnodes, macrophage activation, chemokine expression and chemokinereceptor expression, immunohistochemistry is performed on formalin-fixedparaffin-embedded tissues and/or frozen tissue sections (Mansfield K. G.et al. Am J Pathol 2001, 159:693-702). Tissue sections are cut at 5 μmand immunostained using an avidin-biotin-horseradish peroxidase complextechnique with a diaminobenzidine (DAB) chromogen. Sections are stainedfor the T cell markers CD3 (1.43 μg/ml, DAKO, Carpenteria, Calif.), CD8,CD4, the B cell marker CD20, macrophage markers CD68 and CD163, the MHCII molecule HLA-DR (clone CR3/43, 1.0 μg/ml, DAKO, Carpenteria, Calif.),the α-chemokine receptor CXCR3 (Clone 1C6, 0.33 μg/ml, BD Pharmingen,San Diego, Calif.), and the α-chemokine, monokine induced by INF-γ (MIG,1.0 μg/ml, R&D Systems, Minneapolis, Minn.). Sections are examined withan Olympus Vanox-S AHBS microscope interfaced with a Leica QWin imageanalysis system (Leica Imaging Systems, Cambridge, UK) via a DEI 750charge-coupled device camera (Optronics, Goleta, Calif.). Images of10-20 random fields from each lymph node for each marker are captured at400× magnification. Based on DAB chromogen staining, the area per lymphnode that stains positive is used to calculate the percent area that waspositive for each marker. This technique allows the quantitativeanalysis of immunomorphologic alterations in lymph node structure.

Example 43: Mab 1567-Mediated Lymphocyte Recruitment in a DelayedHypersensitivity Model in Non-Human Primates

The effect of Mab1567 on lymphocyte recruitment in a cutaneous delayedtype hypersensitivity model is evaluated. Preliminary toxicology studiesin the same non-human primate are also performed. As one example,studies on cynomolgus macaques are described below.

A cutaneous delayed type hypersensitivity model is used to examine thedose response to Mab1567 on the temporal recruitment of lymphocytes andmacrophages. Briefly, animals are sensitized to mycobacterial antigensby intradermal injection of Complete Freunds Adjuvant 5-6 months priorto the initiation of the study (Silber A. et al. J Clin Invest 1994,93:1554-1563). Following administration of the test or control antibodyanimals receive 0.1 ml of tuberculin made from standardized filtrates ofMycobacterium tuberculosis (Coopers Animal Health). Multiple intradermalinjections are given along the dorsum and sites are biopsied underanesthesia at 0, 4, 8, 24, 48, 72, 96, 144, 168 and 264 hours afterinjection. Biopsies are split and snap frozen or fixed in 10% neutralbuffered formalin for immunohistochemical analysis and quantitativeimage analysis. A total of four animals are evaluated with animalsrandomly receiving control or test antibody followed by a 1 monthwashout period and cross over to the remaining antibody. The dose to beevaluated is dependant on results of in vivo depletion studies describedabove.

Example 44: In Vivo Toxicology Studies

This protocol is designed to evaluate the toxicity (if any) associatedwith the administration of Mab1567, as well as the efficacy ofCD4⁺/CCR4⁺ depletion from lymph nodes and bone marrow. A weekly dosingregimen is adapted but the specific timing interval depends on theimmunodepletion studies described above. In this study, four animals aregiven weekly doses of circa 15 mg/kg. At the completion of the dosingschedule, lymph node and BM specimens are obtained and analyzed by flowcytometry for the presence of CD4⁺/CCR4⁺ cells. Two animals are examined22 days after the last dose and the other two are examined at 36 days.One control animal is euthanized and evaluated on day 22 and 36.

All animals used in the in vivo studies are evaluated daily for symptomsof toxicity by physical examination, which includes assessment of bodytemperature, weight loss, skin manifestations such as rashes andinfections (due to critical role of skin homing CD4⁺/CCR4⁺ T cells),routine blood chemistry, and urinalysis. Two of the four animals in themultiple high-dose study are euthanized at the end of the study at 22days after final dosing. At necropsy body tissues are thoroughlyexamined for evidences of toxicity and tissue damage. In addition tohistologic analysis, lymph nodes, bone marrow, spleen, thymus andgastrointestinal tissue are obtained for lymphocyte isolation andanalysis. Ex vivo pulmonary lavage is performed and cells are similarlyisolated for FACS analysis. Finally immunohistochemistry withquantitative image analysis is performed to further characterizeimmunomorphologic changes.

It is expected that Mab1567 shows cross-reactivity against cynomolgusmacaque CCR4 as it does with rhesus macaque CCR4 (FIG. 12). It is alsoexpected that immunodepletion of CD4⁺/CCR4⁺ memory T-cells occurs. It isalso possible that immunodepletion of Tregs occurs. Minimalimmunogenicity of Mab1567 is expected because amino acid sequenceanalysis of the IgG heavy chain constant region locus has shown that thehomology between these two non-human primates and humans is very high(>95%)^(115,116). Likewise, V_(kappa) and V_(lambda) variable regiongenes in macaques show 85 to 98% identify with the corresponding humanvariable region genes (Palmisano G. L. et al. Clin Exp Immunol 2004,135:259-266; Hendricks E. E. et al. J Infect Dis 2004, 189:1714-1720).This degree of homology is indistinguishable from that observed between,for example, members of human immunoglobulins from the same gene family.However, this degree of similarity is formally tested by ELISA onanti-CCR4 Mab (anti-idiotype Ab response) verses isotype matched humanIgG1 Mab (anti-isotype Ab response) coated plates using serum recoveredfrom the animals.

Example 45: GMP Cell Bank and Manufacturing

Biovest is contracted to establish and perform safety testing on ourcGMP Master Cell Banks. This service includes establishment of mastercell bank (MBC) of 100 vials and master working cell banks (MWCB). cGMPtesting per cell line is performed for mycoplasma, sterility,bacteriostasis/Fungistasis, advantitious virus and other virus testingBiovest also produces and purifies 5 grams cGMP Mab1567 for thepre-clinical tissue cross-reactivity and pharmacokinetic and toxicologystudies in non-human primates and for the anticipated phase I/IIclinical trial. The bulk purified material is similarly tested as aboveCharles River Laboratories is also contracted to perform stabilitytesting as well as sterile filling of the product vials.

Example 46: Pharmacokinetic and Toxicology Studies in the CynomolgusMacaque

These studies are performed under contact with Aptuit (formerlyQuintiles Edinburgh) using the cGMP Mab1567. These studies include asingle dose acute toxicology study in the rat with a 14 day recoveryPrimate cyclic dose range and MTD dose studies are also performed.Importantly, a 7 cycle repeat dose toxicology study with toxicokineticsampling with 28 days of recovery is conducted. This study is conductedin anticipation of the design of a repeat dosing phase II study. Indeed,several of the monoclonal antibodies in the clinic today for thetreatment of non-Hodgkin's lymphoma (Ritthximab, Campath) use multipledosing (e.g. 5 doses) per cycle. A seven cycle study is conducted toprovide a safety window for the anticipated design of our phase II study(e.g. five weekly doses per cycle).

Example 47: Full Human and Cynomolgus Macaque Tissue Cross-ReactivityStudy with cGMP Mab1567

The studies are performed under GLP compliance. For the human studies, afull human tissue panel (up to 37 human tissues from each of threeunrelated donors) is used with two concentrations of Mab1567 and onenegative control article. For the macaque studies, up to 36 tissues fromeach of two donors are tested with two concentrations of the anti-CCR4Mab and one negative control article. The stained tissue slides areevaluated by a board certified veterinary pathologist and reviewed by asecond veterinary pathologist with similar qualifications

Example 48: Alanine-Scanning Analysis of Functional Residues in the CDRsof Humanized 1567 for Binding to Human CCR4 on Cell Surface

FIG. 17 depicts a graph of percent wild type (WT) binding to 30 aminoacids in the six CDRs of Ab 1567 were mutated to alanine. The humanized1567 scFv-Fc construct (pcDNA3.1-scFv-Fc) was used as the template formutagenesis using QuikChange method (Stratagene). Each mutant wasexpressed and purified as scFv-Fc antibody fragment. Their bindingactivity for CCR4+ Mac-1 cells was analyzed by FACS at variousconcentrations (3-fold serial dilution from 100 ug/mL, total 8concentrations). Mean fluorescence intensity (MFI) of each mutant wasnormalized against wild type humanized 1567-scFv-Fc (100%). % binding ofeach mutant at concentration of 3.7 μg/mL was shown in the figure.Thirteen mutants (highlighted in pink) reduced binding to CCR4 greaterthan 50%. These residues are randomized to generate a library foraffinity maturation of 1567.

The preceding study determined, in part, the following consensussequences. The heavy chain CDRs of the huCCR4 antibody have thefollowing consensus sequences, wherein X is meant to denote any natural,unnatural amino acid or amino acid analogue. For example, X is analanine. In certain embodiments of the invention the heavy chain CDRs ofthe huCCR4 antibody have the following sequences: GYTFASYY (SEQ IDNO:5); WINXXNXNXKYNEKFKG (SEQ ID NO: 11); and SXYXXPLDX (SEQ ID NO: 12).The light chain CDRs of the huCCR4 antibody have the following consensussequences, wherein X is meant to denote any natural, unnatural aminoacid or amino acid analogue. For example, X is an alanine. In certainembodiments of the invention the light chain CDRs of the huCCR4 antibodyhave the following sequences: KSSQSXLYSXXXXNYLA (SEQ ID NO: 13); WASXXES(SEQ ID NO: 14); and HQYLXXYT (SEQ ID NO: 15).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of depleting CCR4+ regulatory T-cells(Tregs) in a human subject comprising administering to the subject ahumanized anti-CCR4 antibody having: a heavy chain with three CDRscomprising the amino acid sequences GYTFASYY (SEQ ID NO:5),WINPGNVNTKYNEKFKG (SEQ ID NO: 6); and STYYRPLDY (SEQ ID NO: 7)respectively, and a light chain with three CDRs comprising the aminoacid sequences KSSQSILYSSNQKNYLA (SEQ ID NO: 8); WASTRES (SEQ ID NO: 9);and HQYLSSYT (SEQ ID NO: 10) respectively; and wherein the antibody hasan IgG1 heavy chain constant region, wherein the subject has ahematologic cancer.
 2. The method of claim 1, wherein said hematologiccancer is cutaneous T-cell Lymphoma (CTCL), mycosis fungoides (MF),primary cutaneous anaplastic large cell Lymphoma (cutaneous ALCL),Sezary syndrome, or adult T cell Leukemia/Lymphoma (ATLL).
 3. A methodof depleting CCR4+ regulatory T-cells (Tregs) in a human subjectcomprising administering to the subject a humanized anti-CCR4 antibodyhaving: a heavy chain with three CDRs comprising the amino acidsequences GYTFASYY (SEQ ID NO:5), WINPGNVNTKYNEKFKG (SEQ ID NO: 6); andSTYYRPLDY (SEQ ID NO: 7) respectively, and a light chain with three CDRscomprising the amino acid sequences KSSQSILYSSNQKNYLA (SEQ ID NO: 8);WASTRES (SEQ ID NO: 9); and HQYLSSYT (SEQ ID NO: 10) respectively; andwherein the antibody comprises a modified glycosylation of the Fcregion, wherein the subject has a hematologic cancer.
 4. A method ofdepleting CCR4+ regulatory T-cells (Tregs) in a human subject comprisingadministering to the subject a humanized anti-CCR4 antibody having: aheavy chain with three CDRs comprising the amino acid sequences GYTFASYY(SEQ ID NO:5), WINPGNVNTKYNEKFKG (SEQ ID NO: 6); and STYYRPLDY (SEQ IDNO: 7) respectively, and a light chain with three CDRs comprising theamino acid sequences KSSQSILYSSNQKNYLA (SEQ ID NO: 8); WASTRES (SEQ IDNO: 9); and HQYLSSYT (SEQ ID NO: 10) respectively; and wherein theantibody is cross-reactive for CCR4 from cynomolgus and/or rhesusmacaques, wherein the subject has a hematologic cancer.
 5. The method ofclaim 3 or 4, wherein said hematologic cancer is cutaneous T-cellLymphoma (CTCL), mycosis fungoides (MF), primary cutaneous anaplasticlarge cell Lymphoma (cutaneous ALCL), Sezary syndrome, or adult T cellLeukemia/Lymphoma (ATLL).